Colorectal cancer recurrence gene expression signature

ABSTRACT

Disclosed are gene signatures that may be used to predict the recurrence of colorectal cancer in a human patient. These signatures can be used to determine when to treat a patient with post-operative adjuvant chemotherapy, i.e., when a high risk of colorectal cancer recurrence is predicted.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No. 61/859,959, filed Jul. 30, 2013, which is hereby incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under Grant No. U01 CA 157960 awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

Colon cancers that are confined within the wall of the colon are often curable with surgery. However, colon cancers that have spread widely around the body are usually not curable and management then focuses on extending the person's life via chemotherapy and improving quality of life. Survival rates for early stage detection is about 5 times that of late stage cancers. For example, patients with a tumor that has not breached the muscularis mucosa (TNM stage Tis, N0, M0) have an average 5-year survival of 100%, while those with an invasive cancer, i.e. T1 (within the submucosal layer) or T2 (within the muscular layer) cancer have an average 5-year survival of approximately 90%. Those with a more invasive tumor, yet without node involvement (T3-4, N0, M0) have an average 5-year survival of approximately 70%. Patients with positive regional lymph nodes (any T, N1-3, M0) have an average 5-year survival of approximately 40%, while those with distant metastases (any T, any N, M1) have an average 5-year survival of approximately 5%. Moreover, more than 50% of patients experience recurrence of the disease after initial treatment of colorectal cancer. Therefore, there is a need in the art for methods that can predict colorectal cancer recurrence, metastasis, and overall survival.

SUMMARY

Disclosed are gene signatures that may be used to predict the recurrence of colorectal cancer in a human patient. A dominant pattern of intrinsic gene expression in colon cancer (referred to herein as “PC1 signature” or “CRC signature”) has been shown to be tightly correlated with a group of genes associated with epithelial-mesenchymal transition (referred to herein as “EMT signature”). There is a 92% correlation (85% r-squared) between the two signatures in a cohort of 326 colorectal cancer tissues. However, as disclosed herein, that the difference between these two scores is much more predictive of metastasis and overall survival than either the CRC signature or EMT signature.

Therefore, disclosed is a method for predicting the recurrence of colorectal cancer in a human patient that involves assaying colorectal cells obtained from the human patient for the expression level of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 115, 116, 117, 118, 119, 120, 121, 122, 123, or more genes listed in TABLE 2A, or their corresponding expression products, and 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 111, 112, 113, 114, 115, 116, 117, 118, or more genes listed in TABLE 2B, or their corresponding expression products, and using normalized values of the expression levels to calculate a CRC signature score. For example, in some embodiments, increased expression of the genes listed in TABLE 2A, or their corresponding expression products, increases the CRC score; and increased expression of the genes listed in TABLE 2B, or their corresponding products, decreases the CRC score.

The method further comprises assaying colorectal cells obtained from the human patient for the expression levels of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 141, 142, 143, 144, 145, 146, 147, 148, or more genes listed in TABLE 1A, or their corresponding expression products, and 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, or more genes listed TABLE 1B, or their corresponding expression products, and using normalized values of the expression levels to calculate an EMT signature score. In some embodiments, gene expression values are first compared to control values to derive differential expression values that are then used to calculate signature scores. For example, in some embodiments, increased expression of the genes listed in TABLE 1A, or their corresponding expression products, increases the EMT score; and increased expression of the genes listed in TABLE 1B, or their corresponding products, decreases the EMT score.

The expression levels for each assayed gene are preferably normalized, such as by quantile normalization, to compensate for differences in sample preparation and measurement techniques. Once normalized, each gene expression value can be given equal weight in an algorithm that calculates each signature score. However, in some cases, different weighting coefficients are assigned to each gene based on multivariate analysis of the gene signature.

The difference between the CRC signature score and the EMT signature score can then be used to arrive at a Recurrence Signature Score (also referred to herein as “ΔPC1.EMT score”) that predicts risk of colorectal cancer recurrence. In some embodiments, the higher the Recurrence Signature Score, the higher the risk of colorectal cancer recurrence.

Also disclosed is a method for analyzing a colorectal cancer tissue sample to determine adjuvant chemotherapy is needed to prevent colorectal cancer recurrence in a human patient that involves first assaying colorectal cells obtained from the human patient for normalized expression values of ten (10) or more genes listed in TABLE 1A, ten (10) or more genes listed in TABLE 1B, ten (10) or more genes listed in TABLE 2A, and ten (10) or more genes listed in TABLE 2B. The method then involves inputting the normalized expression values into a computer programmed to execute an algorithm to convert the normalized expression values to a Recurrence Signature Score indicative of a likelihood of the risk of colorectal cancer recurrence, wherein the algorithm gives reduced weight to the normalized expression values for genes that are listed in more than one of TABLE 1A, TABLE 1B, TABLE 1C, and TABLE 1D.

In some embodiments, the method further involves displaying or outputting to a user, user interface device, computer readable storage medium, or local or remote computer system the calculated risk of colorectal cancer recurrence.

Importantly, the disclosed Recurrence Signature Score may be used to identify patients who may not need adjuvant chemotherapy. Currently Dukes B (stage II) CRC is generally treated by surgical resection alone whereas Dukes C (stage III) CRC is treated with 6 months of post-operative adjuvant chemotherapy. Therefore, the disclosed Recurrence Signature Score may be used to discern a population of stage II CRC patients who might benefit from adjuvant chemotherapy and a population of stage III CRC patients who may not benefit from adjuvant chemotherapy. By using the disclosed Recurrence Signature Score, one can avoid giving chemotherapy to a portion of stage III patients and instead deliver adjuvant therapy selectively to those patients who might actually derive benefit. For example, 54% of people are cured with surgical resection alone in stage III CRC when >10 lymph nodes are involved with metastatic cancer. Adjuvant chemotherapy, while effective, only cures about 14% of these patients; thus, 100 patients are treated to help only 14. The disclosed Recurrence Signature Score can be used to identify the 14% of patients who might actually benefit from adjuvant chemotherapy. For stage 2 patients, approximately 87% of patients are cured with surgery alone; however, it is estimated that 2-10% additional patients might benefit from adjuvant therapy. The disclosed Recurrence Signature Score can be used to identify which of the stage 2 patients might actually benefit from adjuvant chemotherapy. Patients undergoing liver resection for metastatic disease can also benefit from adjuvant chemotherapy. Again, the disclosed Recurrence Signature Score predicting further metastasis and survival can be used to determine which patients might actually benefit from adjuvant chemotherapy following resection. For example, the chemotherapy comprises a 5-fluorouracil (5-FU) therapy.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A to 1D show correlation of PC1, EMT and ΔPC1.EMT scores with each other and with stage, metastasis on Moffitt468 CRC dataset. FIG. 1A is a graph of PC1 scores as a function of EMT scores. PC1 vs. EMT shows strong correlation but metastatic tumor (∘) and primary tumors with metastatic potential (▪) displayed higher PC1 scores than EMT scores as indicated by two separate regression lines. Tumors with “d_meta” are defined as both primary tumors with evidence of synchronous metastasis [stage 4] as well as sampled metastatic lesions. FIGS. 1B and 1C are graphs of ΔPC1.EMT scores as a function of PC1 scores (FIG. 1B) or EMT scores (FIG. 1C). ΔPC1.EMT outperformed EMT and PC1 in predicting metastasis. While solid lines represent median lines, the dash line (EMT=0) is the dividing line as defined (EMT<0, non-EMT epithelial-like; EMT>0, EMT mesenchymal-like). Metastatic lesions (M) (n=101) shown as open circles (∘). Primary tumors (P) with d_meta (n=60) shown as filled squares (▪). Regression line for M and P with d_meta (n=161) shown. Primary tumors without d_meta is shown as plus symbol (+) with a regression line (n=307) (below regression line for M and P with d_meta). FIG. 1D shows EMT score (left), PC1 score (middle) and ΔPC1.EMT score (right) for Stage 1, Stage 2, Stage 3, Stage 4, and Metastatic lesions. ΔPC1.EMT outperformed PCT and EMT in progressively deciphering metastatic potential of primary CRCs. Six samples that lack stage information were removed.

FIGS. 2A to 2C are Kaplan Meier survival graphs of quartile scores on Moffitt468 showing that a higher ΔPC1.EMT predicted poorer overall survival (OS) for all 468 patients (FIG. 2A), for 61 MSI patients (FIG. 2B) and for 407 MSS patients (FIG. 2C).

FIGS. 3A to 3I show that ΔPC1.EMT appears to measure “non-EMT” components of metastasis in addition to EMT components, improving its capacity to predict outcomes. FIGS. 3A to 3D are graph showing ΔPC1.EMT score (in quartiles) remarkably improved the trends (relative to PC1 and EMT) for identifying tumors harboring APC (truncated, FIG. 3A) and BRAF (V600E, FIG. 3B) mutations, and for tumors identified as MSI-H (FIG. 3C) and Stage 4 (FIG. 3D). FIGS. 3E to 3H are graph showing ΔPC1.EMT and EMT trended in opposite directions of the distant metastasis rate in the subgroups of combined mutations KRAS&TP53 (FIG. 3F) or BRAF&TP53 (FIG. 3E) as well as in MSI-H (FIG. 3G) and Stage 1 (FIG. 3H) cases. EMT (▴), PC1 (▪), ΔPC1-EMT (●). FIG. 3I shows weighted analysis of genes contributing to PC1 and EMT vs. ΔPC1.EMT signatures on additional five datasets, suggesting that ΔPC1.EMT is represented by more epithelial (non-EMT) than mesenchymal (EMT) components when compared with the other two scores. The genes that were the most changed from EMT or PC1 to ΔPC1.EMT are shown.

FIGS. 4A to 4F are forest plot summaries of OS Stages 1-3 (FIG. 4A), OS Stage 2 (FIG. 4C), OS Stage 3 (FIG. 4E), RFS Stages 1-3 (FIG. 4B), RFS Stage 2 (FIG. 4D), and RFS Stage 3 (FIG. 4F) analyses of EMT, PC1 and ΔPC1.EMT scores on PETACC, ALMAC, LNCC, GSE14333 and GEO41258 data sets.

FIGS. 5A and 5B are hierarchical cluster analyses using multiple datasets, showing areas of strong overlap in gene expression accounting for the high correlation of EMT to PC1, but also showing more isolated, non-overlapped genes not strongly clustered together suggesting the potential for ΔPC1.EMT to improve outcome.

FIGS. 6A to 6F are pairs plots of the signature scores in PETACC (FIG. 6A), ALAMC (FIG. 6B), French (FIG. 6C), GSE14333 (FIG. 6D), GEO41258 (FIG. 6E), and TCGA (FIG. 6F) datasets.

FIGS. 7A to 7F show gene clusters for PETACC (FIG. 7A), ALAMC (FIG. 7B), French (FIG. 7C), GSE14333 (FIG. 7D), GEO41258 (FIG. 7E), and TCGA (FIG. 7F) datasets.

FIGS. 8A and 8B are boxplot of the PC1.EMT score intensities for APC detrimental mutations or wild-type analyzed by Wilcoxon Rank test (FIG. 8A, p=0.1644) or Kruskal-Wallis Rank test (FIG. 8B, p=0.0181).

FIG. 9A to 9D are plots of AHNAK2 gene log intensities as a function of PC1.EMT score intensities in PETACC (FIG. 9A), French (FIG. 9B), GEO41258 (FIG. 9C), and ALAMC (FIG. 9D) datasets. Example of gene positively correlating with the score

FIG. 10A to 10D are plots of CDX1 gene log intensities as a function of PC1.EMT score intensities in PETACC (FIG. 10A), French (FIG. 10B), GEO41258 (FIG. 10C), and ALAMC (FIG. 10D) datasets. Example of gene negatively correlating with the score FIG. 11 is a forest plot showing correlation between PC1.EMT score intensity and relevant gene signatures. Only signatures showing an absolute estimated coefficient above 0.3 and an adjusted p value bellow 0.05 were plotted.

FIG. 12A to 12U are boxplots of PC1.EMT score split by the clinico-pathological-molecular features age (FIG. 12A), gender (FIG. 12B), stage (FIG. 12C), T.stage (FIG. 12D), N.stage (FIG. 12E), grade (FIG. 12F), adj. treatment (FIG. 12G), lymphovascular invation LVI (FIG. 12H), perineural invasion PNI (FIG. 12I), bowel obstruction (FIG. 12J), positive margin (FIG. 12K), mucinous (FIG. 12L), tumor site (FIG. 12M), left or right tumor site (FIG. 12N), MSI (FIG. 12O), PIK3Ca (FIG. 12P), TP53 (FIG. 12Q), LOH18 (FIG. 12R), BRAF (FIG. 12S), PIK3Ca (FIG. 12T), and TYMS using the PETACC dataset.

FIG. 13A to 13G are boxplots of PC1.EMT score split by the clinico-pathological-molecular features age (FIG. 13A), gender (FIG. 13B), stage (FIG. 13C), T.stage (FIG. 13D), mucinous (FIG. 13E), tumor site (FIG. 13F), and left or right tumor site (FIG. 13G) using the ALMAC dataset.

FIG. 14A to 14K are boxplots of PC1.EMT score split by the clinico-pathological-molecular features age (FIG. 14A), gender (FIG. 14B), stage (FIG. 14C), KRAS (FIG. 14D), MSI (FIG. 14E), KRAS (FIG. 14F), BRAF (FIG. 14G), TP53 (FIG. 14H), CIMP (FIG. 14I), adj. treatment (FIG. 14J), and CIN (FIG. 14K) using the French dataset.

FIG. 15A to 15D are boxplots of PC1.EMT score split by the clinico-pathological-molecular features age (FIG. 15A), gender (FIG. 15B), Dukes stage (FIG. 15C), and tumor site (FIG. 15D) using the GSE14333 dataset.

FIG. 16A to 16I are boxplots of PC1.EMT score split by the clinico-pathological-molecular features age (FIG. 16A), gender (FIG. 16B), stage (FIG. 16C), T.stage (FIG. 16D), N.stage (FIG. 16E), tumor site (FIG. 16F), left or right tumor site (FIG. 16G), MSI (FIG. 16H), and TP53 (FIG. 16I) using the GEO41258 dataset.

FIGS. 17A to 17L are boxplots of PC1.EMT score split by the clinico-pathological-molecular features age (FIG. 17A), gender (FIG. 17B), stage (FIG. 17C), T.stage (FIG. 17D), lympovascular invation (LVI) (FIG. 17E), perineural invasion (PNI) (FIG. 17F), mucinous (FIG. 17G), tumor site (FIG. 17H), left or right tumor site (FIG. 17I), MSI (FIG. 17J), KRAS (FIG. 17K), and BRAF (FIG. 17J) using the TCGA dataset.

FIG. 18A to 18B are boxplot of the PC1.EMT score split by chromosomal instability (CIN) status in TCGA (FIG. 18A) and French (FIG. 18B) datasets.

FIGS. 19A to 19E are show correlations between EMT, PC1 and ΔPC1.EMT scores on the datasets PETACC3 (FIG. 19A), ALMAC (FIG. 19B), GSE14333 (FIG. 19C), GEO41258 (FIG. 19D), and LNCC (FIG. 19E).

FIG. 20 is a plot showing correlation between ΔPC1.EMT and BRAF scores on PETACC dataset (n=752).

FIGS. 21A to 21H show results of OS (FIGS. 21A, 21C, 21E, 21G) and RFS (FIGS. 21B, 21D, 21F, 21H) univariate analysis of ΔPC1.EMT and other 10 known prognostic signatures on the datasets PETACC3 (FIGS. 21A, 21B), ALMAC (FIGS. 21C, 21D), GEO41258 (FIGS. 21E, 21F), LNCC (FIG. 21G), and GSE14333 (FIG. 21H), (significant prognostic values are highlighted by red and blue colors).

DETAILED DESCRIPTION

A dominant pattern of intrinsic gene expression in colon cancer (referred to herein as “PC1 signature” score or “CRC signature” score) is tightly correlated with a group of genes associated with epithelial-mesenchymal transition (referred to herein as “EMT signature” score) (Loboda A, et al. BMC Med Genomics. 2011 4:9). There is a 92% correlation (85% r-squared) between the two signatures in a cohort of 326 colorectal cancer tissues. As disclosed herein, an independent analysis of a subset of 468 of the tissues (which was very strongly validated on the 1563 independent patients that were not part of the subset), demonstrated that the difference between these two scores was much more predictive of metastasis and overall survival than either of the two original signatures, and especially much more statistically significant than the EMT-lung derived signature. This suggests that the 15% (100%−85%) of unexplained variability between the two gene signatures holds the key for predicting metastasis and poor overall survival. Of further surprise, the difference score was significantly positively associated with the EMT signature itself (typically one would expect a negative association between the difference score and the signature being subtracted off in order to obtain it). Liver metastatic tissues were also found to be highly associated with this difference score. However, similar findings were seen when only primary tissue samples were studied, lending further credibility to this signature difference as predictive of distant metastasis and overall survival. Thus, the disclosed “ΔPC1.EMT” score is also referred to herein as a “Recurrence Signature Score” since it can be used to predict the recurrence of colorectal cancer and overall survival.

Methods of “determining gene expression levels” include methods that quantify levels of gene transcripts as well as methods that determine whether a gene of interest is expressed at all. A measured expression level may be expressed as any quantitative value, for example, a fold-change in expression, up or down, relative to a control gene or relative to the same gene in another sample, or a log ratio of expression, or any visual representation thereof, such as, for example, a “heatmap” where a color intensity is representative of the amount of gene expression detected. Exemplary methods for detecting the level of expression of a gene include, but are not limited to, Northern blotting, dot or slot blots, reporter gene matrix, nuclease protection, RT-PCR, microarray profiling, differential display, 2D gel electrophoresis, SELDI-TOF, ICAT, enzyme assay, antibody assay, and MNAzyme-based detection methods. Optionally a gene whose level of expression is to be detected may be amplified, for example by methods that may include one or more of: polymerase chain reaction (PCR), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), or reverse transcription polymerase chain reaction (RT-PCR).

A number of suitable high throughput formats exist for evaluating expression patterns and profiles of the disclosed genes. Numerous technological platforms for performing high throughput expression analysis are known. Generally, such methods involve a logical or physical array of either the subject samples, the biomarkers, or both. Common array formats include both liquid and solid phase arrays. For example, assays employing liquid phase arrays, e.g., for hybridization of nucleic acids, binding of antibodies or other receptors to ligand, etc., can be performed in multiwell or microtiter plates. Microtiter plates with 96, 384 or 1536 wells are widely available, and even higher numbers of wells, e.g., 3456 and 9600 can be used. In general, the choice of microtiter plates is determined by the methods and equipment, e.g., robotic handling and loading systems, used for sample preparation and analysis. Exemplary systems include, e.g., xMAP® technology from Luminex (Austin, Tex.), the SECTOR® Imager with MULTI-ARRAY® and MULTI-SPOT®) technologies from Meso Scale Discovery (Gaithersburg, Md.), the ORCA™ system from Beckman-Coulter, Inc. (Fullerton, Calif.) and the ZYMATE™ systems from Zymark Corporation (Hopkinton, Mass.), miRCURY LNA™ microRNA Arrays (Exiqon, Woburn, Mass.).

Alternatively, a variety of solid phase arrays can favorably be employed to determine expression patterns in the context of the disclosed methods, assays and kits. Exemplary formats include membrane or filter arrays (e.g., nitrocellulose, nylon), pin arrays, and bead arrays (e.g., in a liquid “slurry”). Typically, probes corresponding to nucleic acid or protein reagents that specifically interact with (e.g., hybridize to or bind to) an expression product corresponding to a member of the candidate library, are immobilized, for example by direct or indirect cross-linking, to the solid support. Essentially any solid support capable of withstanding the reagents and conditions necessary for performing the particular expression assay can be utilized. For example, functionalized glass, silicon, silicon dioxide, modified silicon, any of a variety of polymers, such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinations thereof can all serve as the substrate for a solid phase array.

In one embodiment, the array is a “chip” composed, e.g., of one of the above-specified materials. Polynucleotide probes, e.g., RNA or DNA, such as cDNA, synthetic oligonucleotides, and the like, or binding proteins such as antibodies or antigen-binding fragments or derivatives thereof, that specifically interact with expression products of individual components of the candidate library are affixed to the chip in a logically ordered manner, i.e., in an array. In addition, any molecule with a specific affinity for either the sense or anti-sense sequence of the marker nucleotide sequence (depending on the design of the sample labeling), can be fixed to the array surface without loss of specific affinity for the marker and can be obtained and produced for array production, for example, proteins that specifically recognize the specific nucleic acid sequence of the marker, ribozymes, peptide nucleic acids (PNA), or other chemicals or molecules with specific affinity.

Microarray expression may be detected by scanning the microarray with a variety of laser or CCD-based scanners, and extracting features with numerous software packages, for example, IMAGENE™ (Biodiscovery), Feature Extraction Software (Agilent), SCANLYZE™ (Stanford Univ., Stanford, Calif.), GENEPIX™ (Axon Instruments).

In some embodiments, the gene expression values involve numerous data points that are best managed and stored in a computer readable form. Prior to analysis, the data in each dataset can be collected by measuring expression values for each gene, usually in duplicate or triplicate or in multiple replicates. The data may be manipulated, for example raw data may be transformed using standard curves, and the average of replicate measurements used to calculate the average and standard deviation for each gene expression value. These values may be transformed before being used in the models, e.g. log-transformed, Box-Cox transformed, etc.

The disclosed signature scores (CRC signature score and/or EMT signature score) can be determined using standard statistical methods. In some embodiments, the signature score is a regression value. For example, gene expression values (e.g., differential values from controls) may be analyzed by multivariate regression analysis (e.g., determined by linear regression) or principal component analysis to derive a signature score.

In some embodiments, the gene expression values are analyzed by principal component analysis (PCA) to derive the signature scores. PCA is a mathematical procedure that uses an orthogonal transformation to convert a set of observations of possibly correlated variables into a set of values of linearly uncorrelated variables called principal components. The number of principal components is less than or equal to the number of original variables. This transformation is defined in such a way that the first principal component has the largest possible variance (that is, accounts for as much of the variability in the data as possible), and each succeeding component in turn has the highest variance possible under the constraint that it be orthogonal to (i.e., uncorrelated with) the preceding components. When used in the disclosed methods, a PCA score can be a numeric value that summarizes the gene expression of the entire panel (e.g., Tables 4A and/or 4B for CRC signature score) for that patient's biological sample. Therefore, in these embodiments, a “high” signature score (e.g., high CRC signature score) may be a PCA score above the median value, and a “low” risk score (e.g., low CRC signature score) may be a PCA score below the median value.

PCA can be used to reduce gene expression values into a small set of uncorrelated principal components based on their ability to account for variation. The first principal component (1st PCA), as it accounts for the largest variability in the data, can be to represent the overall expression level for the set of genes.

In some cases, the signature scores are calculated as a weighted average expression among the normalized expression values, e.g., by the formula Σw_(i)x_(i), where x_(i) represents gene i expression level, w_(i) is the corresponding weight (loading coefficient) with Σw² _(i)=1, and the w_(i) values maximize the variance of Σw_(i)x_(i).

As will be appreciated by those of skill in the art, a number of quantitative criteria can be used to communicate the performance of the comparisons made between a test marker profile and reference marker profiles. These include area under the curve (AUC), hazard ratio (HR), relative risk (RR), reclassification, positive predictive value (PPV), negative predictive value (NPV), accuracy, sensitivity and specificity, Net reclassification Index, Clinical Net reclassification Index. In addition, other constructs such a receiver operator curves (ROC) can be used to evaluate analytical process performance.

Table 1A lists the 149 gene markers that were found to be up-regulated in lung cancer cell lines that were classified as mesenchymal cell-like, as compared to the lung cancer cell lines that were classified as epithelial cell-like, and were also found to be down-regulated in the lung tumor cell lines that were classified as epithelial cell-like as compared to the lung cancer cell lines that were classified as mesenchymal cell-like. Table 1A provides for each of the 149 gene markers, the gene symbol; the Genbank reference number for each gene symbol as of Oct. 1, 2010, each of which is hereby incorporated herein by reference; and the SEQ ID NO: corresponding to an exemplary 60-mer sequence that corresponds to a portion of the corresponding cDNA, which may be used as a probe.

TABLE 1A 149 EMT Signature Genes: Up-Regulated Arm. Gene Genbank Ref. SEQ ID Symbol Number NO: FAM171A1 AY683003 1 ZCCHC24 BC028617 2 GLIPR2 AK091288 3 TMSB15A BG471140 4 COL12A1 NM_004370 5 LOX NM_002317 6 SPARC AK126525 7 CDH11 D21255 8 ZEB 1 BX647794 9 EML1 NM_001008707 10 ZNF788 AK128700 11 WIPF1 NM_001077269 12 CAP2 NM_006366 13 TGFB2 AB209842 14 DLC1 NM_182643 15 POSTN NM_006475 16 NEGRI NM_173808 17 JAM3 AK027435 18 SRPX BC020684 19 BICC1 NM_001080512 20 HAS2 NM_005328 21 ANTXR1 NM_032208 22 GNB4 NM_021629 23 COL4A1 NM_001845 24 SRGN CD359027 25 SUSD5 NM_015551 26 DI02 NM_013989 27 GLIPR1 NM_006851 28 COL5A1 NM_000093 29 NAP1L3 BC094729 30 RBMS3 BQ214991 31 BVES BC040502 32 SLC47A1 BC010661 33 FGFR1 NM_023110 34 FSTL1 NM_007085 35 FGF2 NM_002006 36 DKK3 NM_015881 37 CMTM3 AK056324 38 PTGIS NM_000961 39 CCL2 BU570769 40 WNT5B BC001749 41 CLDN11 AK098766 42 MAP IB NM_005909 43 IL13RA2 AK308523 44 MSRB3 NM_001031679 45 FAM101B AK093557 46 ZEB2 NM_014795 47 NIDI NM_002508 48 TMEM158 NM_015444 49 ST3GAL2 AK127322 50 FGF5 NM_004464 51 AKAP12 NM_005100 52 GPR176 BC067106 53 PMP22 NM_000304 54 LEPREL1 NM_018192 55 CHN1 NM_001822 56 TTC28 NM_001145418 57 GLT25D2 NM_015101 58 RECK BX648668 59 GREM1 NM_013372 60 C16orf45 AK092923 61 AOX1 LI 1005 62 CTGF NM_001901 63 ANXA6 NM_001155 64 SERPINE1 NM_000602 65 SLC2A3 AB209607 66 ZFPM2 NM_012082 67 FHL1 NM_001159704 68 ATP8B2 NM_020452 69 RBPMS2 AY369207 70 TBXA2R NM_001060 71 COL3A1 NM_000090 72 GPC6 NM_005708 73 AFF3 NM_002285 74 PLAGL1 CR749329 75 LGALS1 BF570935 76 TTLL7 NM_024686 77 COL5A2 NM_000393 78 ANKRD1 NM_014391 79 NRG1 NM_013960 80 POPDC3 NM_022361 81 C1S NM_201442 82 CDH2 NM_001792 83 DOCK10 NM_014689 84 CLIP3 AK094738 85 CDH4 AL834206 86 COL6A1 NM_001848 87 HEG1 NM_020733 88 IGFBP7 BX648756 89 DAB2 NM_001343 90 F2R NM_001992 91 EDIL3 BX648583 92 COL1A2 J03464 93 HTRA1 NM_002775 94 NDN NM_002487 95 BDNF EF689009 96 LHFP NM_005780 97 PRKD1 X75756 98 MMP2 NM_004530 99 UCHL1 AB209038 100 DPYSL3 BC077077 101 RBM24 AL832199 102 DFNA5 AK094714 103 MRAS NM_012219 104 SYDE1 AK128870 105 FLRT2 NM_013231 106 AK5 NM_012093 107 EPDR1 XM_002342700 108 TUB NM_003320 109 SIRPA NM_001040022 110 AXL NM_021913 111 FBN1 NM_000138 112 EVI2A NM_001003927 113 PTX3 NM_002852 114 ADAM23 AK091800 115 PNMA2 NM_007257 116 PDE7B AB209990 117 TCF4 NM_001083962 118 KIRREL AK090554 119 NEXN NM_144573 120 ALPK2 BX647796 121 VIM NM_003380 122 LIX1L AK128733 123 ADAMTS1 NM_006988 124 PAPPA NM_002581 125 ANGPTL2 NM_012098 126 AP1S2 BX647483 127 TUBA1A BI083878 128 LAMA4 NM_001105206 129 EPB41L5 BC054508 130 NAV3 NM_014903 131 ELOVL2 BC050278 132 BNC2 NM_017637 133 GFPT2 BC000012 134 TRPA1 Y10601 135 PRR16 AF242769 136 CYBRD1 NM_024843 137 HS3ST3A1 NM_006042 138 GNG11 BF971151 139 TMEM47 BC039242 140 CPA4 NM_016352 141 ARMCX1 CR933662 142 RFTN1 NM_015150 143 EMP3 BM556279 144 ATP8B3 AK125969 145 FAT4 NM_024582 146 NUDT11 NM_018159 147 PTRF NM_012232 148 TNFRSF19 NM_148957 149

Table 1B lists the 161 gene markers that were found to be down-regulated in the lung tumor cell lines that were classified as mesenchymal cell-like, as compared to the lung cancer cell lines that were classified as epithelial cell-like, and were also found to be up-regulated in the lung cancer cell lines that were classified as epithelial cell-like as compared to the lung cancer cell lines that were classified as mesenchymal cell-like. Table 1B provides for each of the 161 gene markers, the gene symbol; the Genbank reference number for each gene symbol as of Oct. 1, 2010, each of which is hereby incorporated herein by reference; and the SEQ ID NO: corresponding to an exemplary 60-mer sequence that corresponds to a portion of the corresponding cDNA, which may be used as a probe.

TABLE 1B 161 EMT Signature Genes: Down-Regulated Arm. Gene Genbank Ref. SEQ ID Symbol Number NO: PRR15L BC002865 150 TTC39A AB007921 151 ESRP1 NM_017697 152 RBM35B CR607695 153 AGR3 BG540617 154 TMEM125 BC072393 155 KLK8 DQ267420 156 MBNL3 NM_001170704 157 SPRR1B AI541215 158 S100A9 BQ927179 159 TMC5 NM_001105248 160 ELF5 NM_198381 161 ERBB3 NM_001982 162 WDR72 NM_182758 163 FAM84B NM_174911 164 SPRR3 EF553525 165 TMEM30B NM_001017970 166 Clorf210 NM_182517 167 TMPRSS4 NM_019894 168 ERP27 BC030218 169 TTC22 NM_017904 170 CNKSR1 BC012797 171 FGFBP1 NM_005130 172 FUT3 NM_000149 173 GALNT3 NM_004482 174 RAPGEF5 NM_012294 175 MAPK13 AB209586 176 AP1M2 BC005021 177 CDH3 NM_001793 178 PPL NM_002705 179 GCNT3 EF152283 180 EPPK1 AB051895 181 MAL2 NM_052886 182 TMPRSS11E NM_014058 183 LCN2 AK307311 184 ANKRD22 NM_144590 185 POU2F3 AF162715 186 SPINT1 BC018702 187 AQP3 NM_004925 188 GPR110 CR627234 189 FAM84A NM_145175 190 TMPRSS13 NM_001077263 191 GPX2 BE512691 192 WFDC2 BM921431 193 KLK10 NM_002776 194 S100A14 BG674026 195 S100P BG571732 196 FXYD3 BF676327 197 MUC20 XR_078298 198 SPINT2 NM_021102 199 Clorf1l6 NM_023938 200 SPINK5 NM_001127698 201 ANXA9 NMJX568 202 TMC4 NM_001145303 203 SYK NM_003177 204 HOOK1 NM_015888 205 FAM83A DQ280323 206 LCP1 NM_002298 207 HS6ST2 NM_001077188 208 TSPAN1 NM_005727 209 S100A8 BG739729 210 DMKN BC035311 211 GRHL1 NM_198182 212 CKMT1B AK094322 213 ACPP NM_001099 214 PTAFR NM_000952 215 KRT5 M21389 216 DAPP1 NM_014395 217 LAMA3 NM_198129 218 C19orf21 NM_173481 219 SH2D3A AK024368 220 TOX3 AK095095 221 CDH1 NM_004360 222 FA2H NM_024306 223 SPRR1A NM_005987 224 LIPG BC060825 225 CEACAM6 NM_002483 226 PROM2 NM_001165978 227 ITGB6 AL831998 228 OR2A4 BC120953 229 MAP7 NM_003980 230 PPPR14C AF407165 231 PVRL4 NM_030916 232 FBP1 NM_00507 233 FAAH2 NM_174912 234 LAMB3 NM_001017402 235 MPP7 NM_173496 236 ANK3 NM_020987 237 SYT7 NM_004200 238 TRIP29 BX648072 239 TMEM45B AK098106 240 ST14 NM_021978 241 ARHGDIB AK125625 242 HS3ST1 AK096823 243 KLK5 AY359010 244 GJB6 NM_001110219 245 CCDC64B NM_001103175 246 PAK6 AK131522 247 MARVELD3 NM_001017967 248 CLDN7 NM_001307 249 SH3YL1 AK123829 250 SLPI BG483345 251 MB BF670653 252 NPNT NM_001033047 253 Clorf106 NM_001142569 254 DSP NM_004415 255 STEAP4 NM_024636 256 SLC6A14 NM_007231 257 GOLT1A AB075871 258 PKP3 NM_007183 259 SCEL BC047536 260 VTCN1 BX648021 261 SERPEMB5 BX640597 262 DEND2D AL713773 263 PLA2G10 NM_003561 264 SCNN1A AK172792 265 GPR87 NM_023915 266 IRF6 NM_006147 267 CGN BC146657 268 LAMC2 NM_005562 269 RASGEF1B BX648337 270 KRTCAP3 AY358993 271 GRAMD2 BC038451 272 BSPRY NM_017688 273 ATP2C2 AB014603 274 SORBS2 BC069025 275 RAB25 BE612887 276 CLDN4 AK126462 277 EHF NM_012153 278 KRT19 BQ073256 279 CDS1 NM_001263 280 KRT16 NM_005557 281 CNTNAP2 NM_014141 282 MARVELD2 AK055094 283 RASEF NM_152573 284 ΓNPP4B NM_003866 285 OVOL2 AK022284 286 GRHL2 NM_024915 287 BLNK AK225546 288 EPN3 NM_017957 289 ELF3 NM_001114309 290 STX19 NM_001001850 291 B3GNT3 NM_014256 292 FUT1 NM_000148 293 CEACAM5 NM_004363 294 MY05B NM_001080467 295 ARHGAP8 BC059382 296 PRSS8 NM_002773 297 TTC9 NM_015351 298 KLK6 NM_002774 299 IL1RN BC068441 300 FAM110C NM_001077710 301 ALDH3B2 AK092464 302 PRR15 NM_175887 303 DSC2 NM_004949 304 Cllorf52 BC110872 305 ILDR1 BC044240 306 CD24 AK125531 307 CTAGE4 DB515636 308 FGD2 BC023645 309 MYH14 NM_001145809 310

The 60mer sequences provided in Tables 2A and 2B are non-limiting examples of exemplary probes that correspond to a portion of the corresponding cDNA.

A refined set of CRC Signature genes were selected from the about 5000 first principal component (PC1) genes identified by performing Principal Component Analysis (“PCA”) on robust multi-array (RMA)-normalized data obtained from the U133 Plus 2.0 Affymetrix arrays. The RMA-normalized dataset consisted of the 326 CRC tumor profiles. A first principal component (PC1) was selected and for each probe-set. (i.e., gene transcript represented on the array), a Spearman correlation was computed to the PC1. Then, the 200 probe-sets with the highest value of correlation coefficient to PC1 were selected, and the list of unique markers for these probe-sets was used to generate the 124 CRC Signature Mesenchymal marker list shown in Table 2A. Table 2A provides for each of the 124 CRC Signature Mesenchymal markers, the gene symbol the Genbank reference number for each gene symbol as of Oct. 1, 2010, each of which is hereby incorporated herein by reference; and the SEQ ID NO: corresponding to an exemplary 60-mer sequence that corresponds to a portion of the corresponding cDNA, which may be used as a probe.

TABLE 2A 124 CRC Signature Genes: The Mesenchymal or Up-Regulated Arm. Gene Genbank Ref. SEQ ID Symbol Number NO: SPARC AK126525 7 CAP2 NM_006366 13 JAM3 AK027435 18 SRPX BC020684 19 NAP1L3 BC094729 30 CMTM3 AK056324 38 MAP IB NM_005909 43 MSRB3 NM_001031679 45 AKAP12 NM_005100 52 RECK BX648668 59 ZFPM2 NM_012082 67 ATP8B2 NM_020452 69 LGALS1 BF570935 76 HTRA1 NM_002775 94 NDN NM_002487 95 LHFP NM_005780 97 PRKD1 X75756 98 UCHL1 AB209038 100 DPYSL3 BC077077 101 DFNA5 AK094714 103 MRAS NM_012219 104 FLRT2 NM_013231 106 VIM NM_003380 122 LIX1L AK128733 123 AP1S2 BX647483 127 GFPT2 BC000012 134 TRPA1 Y10601 135 GNG11 BF971151 139 ARMCX1 CR933662 142 PTRF NM_012232 148 AEBP1 NM_001129 311 AKT3 NM_005465 312 AMOTL1 NM_130847 313 ANKRD6 NM_014942 314 ARMCX2 NM_014782 315 BASP1 NM_006317 316 BGN NM_001711 317 Clorf54 NM_024579 318 C20orf194 NM_001009984 319 CALD1 NM_004342 320 CCDC80 NM_199511 321 CEP170 NM_001042404 322 CFH NM_000186 323 CFL2 NM_021914 324 COX7A1 NM_001864 325 CRYAB NM_001885 326 DCN NM_001920 327 DNAJB4 NM_007034 328 DZIP1 NM_014934 329 ECM2 NM_001393 330 EFHA2 NM_181723 331 EFS NM_005864 332 EHD3 NM_014600 333 FAM20C NM_020223 334 FBXL7 NM_012304 335 FEZ1 NM_005103 336 FRMD6 NM_001042481 337 GLIS2 NM_032575 338 HECTD2 NM_173497 339 IL1R1 NM_000877 340 KCNE4 NM_080671 341 KIAA1462 NM_020848 342 KLHL5 NM_001007075 343 LAYN NM_178834 344 LDB2 NM_001130834 345 LMCD1 NM_014583 346 LPHN2 NM_012302 347 LZTS1 NM_021020 348 MAF NM_001031804 349 MAGEH1 NM_014061 350 MAP9 NM_001039580 351 MCC NM_001085377 352 MGP NM_000900 353 MLLT11 NM_006818 354 MPDZ NM_003829 355 MSN NM_002444 356 MXRA7 NM_001008528 357 MYH10 NM_005964 358 MY05A NM_000259 359 NNMT NM_006169 360 NR3C1 NM_000176 361 NRP1 NM_001024628 362 NRP2 NM_003872 363 PEA15 NM_003768 364 PFTK1 NM_012395 365 PHLDB2 NM_001134437 366 PKD2 NM_000297 367 PRICKLE1 NM_001144881 368 PTPRM NM_001105244 369 QKI NM_006775 370 RAB31 NM_006868 371 RAB34 NM_001142624 372 RAI14 NM_001145520 373 RASSF8 NM_001164746 374 RGS4 NM_001102445 375 RNF180 NM_001113561 376 SCHIP1 NM_014575 377 SDC2 NM_002998 378 SERPINF1 NM_002615 379 SGCE NM_001099400 380 SGTB NM_019072 381 SLIT2 NM_004787 382 SMARCA1 NM_003069 383 SNAI2 NM_003068 384 SPG20 NM_001142294 385 SRGAP2 NM_001042758 386 STON1 NM_006873 387 SYT11 NM_152280 388 TCEA2 NM_003195 389 TCEAL3 NM_001006933 390 TIMP2 NM_003255 391 TNS1 NM_022648 392 TPST1 NM_003596 393 TRPC1 NM_003304 394 TRPS1 NM_014112 395 TSPYL5 NM_033512 396 TTC7B NM_001010854 397 TUBB6 NM_032525 398 TUSC3 NM_006765 399 UBE2E2 NM_152653 400 WWTR1 NM_001168278 401 ZNF25 NM_145011 402 ZNF532 NM_018181 403 ZNF677 NM_182609 404

Similarly, 200 probe-sets with the most negative correlation coefficient to PC1 were taken, and the corresponding list of 119 unique markers was used to generate the CRC Signature Epithelial marker list shown in Table 21B. Table 2B provides for each of the 119 CRC Signature Epithelial markers, the gene symbol; the Genbank reference number for each gene symbol as of Oct. 1, 2010, each of which is hereby incorporated herein by reference; and the SEQ ID NO: corresponding to an exemplary 60-mer sequence that corresponds to a portion of the corresponding cDNA, which may be used as a probe.

TABLE 2B 119 CRC Signature Genes: The Epithelial or Down-Regulated Arm. Gene Genbank Ref. SEQ ID Symbol Number NO: TMC5 NM_001105248 160 FUT3 NM_000149 173 AP1M2 BC005021 177 FAM84A NM_145175 190 GPX2 BE512691 192 CKMT1B AK094322 213 FA2H NM_024306 223 MAP7 NM_003980 230 ST14 NM_021978 241 MARVELD3 NM_001017967 248 RAB25 BE612887 276 CDS1 NM_001263 280 EPN3 NM_017957 289 MY05B NM_001080467 295 MYH14 NM_001145809 310 ACOT11 NM_015547 405 AGMAT NM_024758 406 ANKS4B NM_145865 407 ATP10B NM_025153 408 AXIN2 NM_004655 409 BCAR3 NM_003567 410 BCL2L14 NM_030766 411 BDH1 NM_004051 412 BRI3BP NM_080626 413 C10orf99 NM_207373 414 C4orf19 NM_001104629 415 C9orf152 NM_001012993 416 C9orf75 NM_001128228 417 C9orf82 NM_001167575 418 CALML4 NM_001031733 419 CAPN5 NM_004055 420 CASP5 NM_001136109 421 CASP6 NM_001226 422 CBLC NM_001130852 423 CC2D1A NM_017721 424 CCL28 NM_148672 425 CDC42EP5 NM_145057 426 CDX1 NM_001804 427 CLDN3 NM_001306 428 CMTM4 NM_178818 429 COR02A NM_003389 430 COX10 NM_001303 431 CYP2J2 NM_000775 432 DAZAP2 NM_001136264 433 DDAH1 NM_001134445 434 DTX2 NM_001102594 435 DUOX2 NM_014080 436 DUOXA2 NM_207581 437 ENTPD5 NM_001249 438 EPB41L4B NM_018424 439 EPHB2 NM_004442 440 EPS8L3 NM_024526 441 ESRRA NM_004451 442 ETHE1 NM_014297 443 EXPH5 NM_001144763 444 F2RL1 NM_005242 445 FAM3D NM_138805 446 FAM83F NM_138435 447 FRAT2 NM_012083 448 FUT2 NM_000511 449 FUT5 NM_002033 450 FUT6 NM_000150 451 GALNT7 NM_017423 452 GMDS NM_001500 453 GPA33 NM_005814 454 GPR35 NM_005301 455 HDHD3 NM_031219 456 HMGA1 NM_002131 457 HNF4A NM_000457 458 HOXB9 NM_024017 459 HSD11B2 NM_000196 460 KALRN NM_001024660 461 KCNE3 NM_005472 462 KCNQ1 NM_000218 463 KIAA0152 NM_014730 464 LENG9 NM_198988 465 LGALS4 NM_006149 466 LRRC31 NM_024727 467 MCCC2 NM_022132 468 MPST NM_001013436 469 MRPS35 NM_021821 470 MUC3B XM_001125753.2 471 MYB NM_001130172 472 MY07B NM_001080527 473 NAT2 NM_000015 474 NOB1 NM_014062 475 NOX1 NM_007052 476 NR1I2 NM_003889 477 PAQR8 NM_133367 478 PI4K2B NM_018323 479 PKP2 NM_001005242 480 PLA2G12A NM_030821 481 PLEKHA6 NM_014935 482 PLS1 NM_001145319 483 PMM2 NM_000303 484 POF1B NM_024921 485 PPP1R1B NM_032192 486 PREP NM_002726 487 RNF186 NM_019062 488 SELENBP1 NM_003944 489 SH3RF2 NM_152550 490 SHH NM_000193 491 SLC12A2 NM_001046 492 SLC27A2 NM_001159629 493 SLC29A2 NM_001532 494 SLC35A3 NM_012243 495 SLC37A1 NM_018964 496 SLC44A4 NM_001178044 497 SLC5A1 NM_000343 498 SLC9A2 NM_003048 499 STRBP NM_001171137 500 SUCLG2 NM_001177599 501 SULT1B1 NM_014465 502 TJP3 NM_014428 503 TMEM54 NM_033504 504 TMPRSS2 NM_001135099 505 TST NM_003312 506 USP54 NM_152586 507 XK NM_021083 508

The markers represented in Tables 2A and 2B are collectively referred to as CRC Signature genes. Markers that are also present in the EMT Signature lists (Tables 1A and 1B) are indicated at the beginning of both Tables 2A and 2B. In total, 30 gene markers listed in Tables 4A are also present in Table 1A, and 15 gene markers listed in Table 21B are also present in Table 1B. The 60mer sequences provided in Tables 2A and 2B3 are non-limiting examples of exemplary probes that correspond to a portion of the corresponding cDNA.

As disclosed herein, the result of subtracting the EMT signature score from its strongly related PC1 signature score produces a best in class “difference score” (ΔPC1.EMT) that is far more predictive of metastasis and outcome than either score alone. Table 3A below lists the genes that are common to both the CRC and EMT signature gene panels. Table 3B lists the genes that do not overlap.

TABLE 3A Overlapping CRC-EMT genes CRC.EMT Up-Regulated CRC.EMT Down-Regulated AKAP12 MAP1B AP1M2 AP1S2 MRAS CDS1 ARMCX1 MSRB3 CKMT1B ATP8B2 NAP1L3 EPN3 CAP2 NDN FA2H CMTM3 PRKD1 FAM84A DFNA5 PTRF FUT3 DPYSL3 RECK GPX2 FLRT2 SERPINF1 MAP7 GFPT2 SPARC MARVELD3 GNG11 SRPX MYH14 HTRA1 TRPA1 MYO5B JAM3 UCHL1 RAB25 LGALS1 VIM ST14 LHFP ZFPM2 TMC5 LIX1L

TABLE 3B Non-overlapping CRC-EMT genes EMT (Lung) UP EMT (Lung) DOWN CRC UP CRC DOWN ADAM23 LOX ACPP KRT16 AEBP1 SPG20 ACOT11 PAQR8 ADAMTS1 MMP2 AGR3 KRT19 AKT3 SRGAP2 AGMAT PI4K2B AFF3 NAV3 ALDH3B2 KRT5 AMOTL1 STON1 ANKS4B PKP2 AK5 NEGR1 ANK3 KRTCAP3 ANKRD6 SYT11 ATP10B PLA2G12A ALPK2 NEXN ANKRD22 LAMA3 ARMCX2 TCEA2 AXIN2 PLEKHA6 ANGPTL2 NID1 ANXA9 LAMB3 BASP1 TCEAL3 BCAR3 PLS1 ANKRD1 NRG1 AQP3 LAMC2 BGN TIMP2 BCL2L14 PMM2 ANTXR1 NUDT11 ARHGAP8 LCN2 C1orf54 TNS1 BDH1 POF1B ANXA6 PAPPA ARHGDIB LCP1 C20orf194 TPST1 BRI3BP PPP1R1B AOX1 PDE7B ATAD4 LIPG CALD1 TRPC1 C10orf99 PREP ATP8B3 PLAGL1 ATP2C2 MAL2 CCDC80 TRPS1 C4orf19 RNF186 AXL PMP22 B3GNT3 MAPK13 CEP170 TSPYL5 C9orf152 SELENBP1 BDNF PNMA2 BLNK MARVELD2 CFH TTC7B C9orf75 SH3RF2 BICC1 POPDC3 BSPRY MB CFL2 TUBB6 C9orf82 SHH BNC2 POSTN C11orf52 MBNL3 COX7A1 TUSC3 CALML4 SLC12A2 BVES PRR16 C19orf21 MPP7 CRYAB UBE2E2 CAPN5 SLC27A2 C10orf38 PTGIS C1orf106 MUC20 DCN WWTR1 CASP5 SLC29A2 C10orf56 PTX3 C1orf116 NPNT DNAJB4 ZNF25 CASP6 SLC35A3 C16orf45 RBM24 C1orf210 OR2A4 DZIP1 ZNF532 CBLC SLC37A1 C1S RBMS3 C1orf34 OVOL2 ECM2 ZNF677 CC2D1A SLC44A4 C9orf19 RBPMS2 CCDC64B PAK6 EFHA2 CCL28 SLC5A1 CCL2 RFTN1 CD24 PKP3 EFS CDC42EP5 SLC9A2 CDH11 SIRPA CDH1 PLA2G10 EHD3 CDX1 STRBP CDH2 SLC2A3 CDH3 POU2F3 FAM20C CLDN3 SUCLG2 CDH4 SLC47A1 CEACAM5 PPL FBXL7 CMTM4 SULT1B1 CHN1 SRGN CEACAM6 PPP1R14C FEZ1 CORO2A TJP3 CLDN11 ST3GAL2 CGN PROM2 FRMD6 COX10 TMEM54 CLIP3 SUSD5 CLDN4 PRR15 GLIS2 CYP2J2 TMPRSS2 COL12A1 SYDE1 CLDN7 PRSS8 HECTD2 DAZAP2 TST COL1A2 TBXA2R CNKSR1 PTAFR IL1R1 DDAH1 USP54 COL3A1 TCF4 CNTNAP2 PVRL4 KCNE4 DTX2 XK COL4A1 TGFB2 CTAGE4 RAPGEF5 KIAA1462 DUOX2 COL5A1 TMEM158 DAPP1 RASEF KLHL5 DUOXA2 COL5A2 TMEM47 DENND2D RASGEF1B LAYN ENTPD5 COL6A1 TMSL8 DMKN RBM35A LDB2 EPB41L4B CPA4 TNFRSF19 DSC2 RBM35B LMCD1 EPHB2 CTGF TTC28 DSP S100A14 LPHN2 EPS8L3 CYBRD1 TTLL7 EHF S100A8 LZTS1 ESRRA DAB2 TUB ELF3 S100A9 MAF ETHE1 DIO2 TUBA1A ELF5 S100P MAGEH1 EXPH5 DKK3 WIPF1 EPPK1 SCEL MAP9 F2RL1 DLC1 WNT5B ERBB3 SCNN1A MCC FAM3D DOCK10 ZEB1 ERP27 SERPINB5 MGP FAM83F EDIL3 ZEB2 FAAH2 SH2D3A MLLT11 FRAT2 ELOVL2 ZNF788 FAM110C SH3YL1 MPDZ FUT2 EML1 FAM83A SLC6A14 MSN FUT4 EMP3 FAM84B SLPI MXRA7 FUT6 EPB41L5 FBP1 SORBS2 MYH10 GALNT7 EPDR1 FGD2 SPINK5 MYO5A GMDS EVI2A FGFBP1 SPINT1 NNMT GPA33 F2R FUT1 SPINT2 NR3C1 GPR35 FAM101B FXYD3 SPRR1A NRP1 HDHD3 FAT4 GALNT3 SPRR1B NRP2 HMGA1 FBN1 GCNT3 SPRR3 PEA15 HNF4A FGF2 GJB6 STEAP4 PFTK1 HOXB9 FGF5 GOLT1A STX19 PHLDB2 HSD11B2 FGFR1 GPR110 SYK PKD2 KALRN FHL1 GPR87 SYT7 PRICKLE1 KCNE3 FSTL1 GRAMD2 TMC4 PTPRM KCNQ1 GLIPR1 GRHL1 TMEM125 QKI KIAA0152 GLT25D2 GRHL2 TMEM30B RAB31 LENG9 GNB4 HOOK1 TMEM45B RAB34 LGALS4 GPC6 HS3ST1 TMPRSS11E RAI14 LRRC31 GPR176 HS6ST2 TMPRSS13 RASSF8 MCCC2 GREM1 IL1RN TMPRSS4 RGS4 MPST HAS2 ILDR1 TOX3 RNF180 MRPS35 HEG1 INPP4B TRIM29 SCHIP1 MUC3B HS3ST3A1 IRF6 TSPAN1 SDC2 MYB IGFBP7 ITGB6 TTC22 SGCE MYO7B IL13RA2 KLK10 TTC9 SGTB NAT2 KIRREL KLK5 VTCN1 SLIT2 NOB1 LAMA4 KLK6 WDR72 SMARCA1 NOX1 LEPREL1 KLK8 WFDC2 SNAI2 NR1I2

In some embodiments of the disclosed methods, a low Recurrence signature score can be an indication of a favorable prognosis for the patient. A favorable prognosis can involve an increased likelihood of survival after treatment with chemotherapy. For example, a favorable prognosis can be a greater than 47%, 48%, 49%, 50%, 60%, 70%, 80%, or 90% chance of survival for at least five years.

Definitions

The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

The term “sample from a subject” refers to a tissue (e.g., tissue biopsy), organ, cell (including a cell maintained in culture), cell lysate (or lysate fraction), biomolecule derived from a cell or cellular material (e.g. a polypeptide or nucleic acid), or body fluid from a subject. Non-limiting examples of body fluids include blood, urine, plasma, serum, tears, lymph, bile, cerebrospinal fluid, interstitial fluid, aqueous or vitreous humor, colostrum, sputum, amniotic fluid, saliva, anal and vaginal secretions, perspiration, semen, transudate, exudate, and synovial fluid.

The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

The term “cancer” or “malignant neoplasm” refers to a cell that displays uncontrolled growth, invasion upon adjacent tissues, and often metastasis to other locations of the body.

The term “metastasis” refers to the spread of malignant tumor cells from one organ or part to another non-adjacent organ or part. Cancer cells can “break away,” “leak,” or “spill” from a primary tumor, enter lymphatic and blood vessels, circulate through the bloodstream, and settle down to grow within normal tissues elsewhere in the body. When tumor cells metastasize, the new tumor is called a secondary or metastatic cancer or tumor.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention.

Accordingly, other embodiments are within the scope of the following claims.

EXAMPLES Example 1: Non-EMT Features Optimize Prediction of Colorectal Cancer Metastasis

Colorectal cancer (CRC) still represents a prognostic challenge because it is difficult to identify which patients will ultimately progress and succumb to their disease. An EMT signature is highly correlated to the first principal component (PC1) of a large CRC gene expression data set (Loboda. A. et al. BMC Med Genomics. 2011 4:9). Both EMT and PC1 were prognostic for survival and recurrence of disease. However, as disclosed herein, the result of subtracting the EMT signature score from its strongly related PC1 signature score produces a best in class “difference score” (ΔPC1.EMT) that is far more predictive of metastasis and outcome than either score alone. This result was highly reproducible on six independent test sets (n>4000 CRC tumors), performing well in Stages 1-3, amongst MSI subtypes, and across multiple mutation-based subclasses. The improved performance of ΔPC1.EMT to predict metastasis appears to be related to its bias to identify epithelial (non-EMT) as well as mesenchymal (EMT) subpopulations, supporting a cooperative model for metastatic progression involving both cell types. While EMT is a dominant differential molecular program of CRC and sufficient to predict outcome, non-EMT features, including epithelial cancer stem cell-related properties, are necessary to optimally predict metastatic potential, and may need to be targeted to overcome distant disease.

The heterogeneity of colorectal cancer makes it difficult to determine which patients will benefit from adjuvant therapy and which patients do not require further therapy beyond surgical resection. To address this problem, several gene expression signatures have been developed to identify molecular subpopulations of human CRC with poor prognosis (Loboda, A. et al. BMC Med Genomics. 2011 4:9; Eschrich, S. et al. J Clin Oncol. 2005 23(15):3526-35; Jorissen, R. N. et al. Clin Cancer Res. 2009 15(24):7642-7651; Sotiriou, C. et al. J Natl Cancer Inst. 2006 98(4):262-72; Farmer, P. et al. Nat Med. 2009 15(1):68-74); Roth, A. D. et al. J Natl Cancer Inst. 2012 104(21):1635-46; Popovici, V. et al. J Clin Oncol. 2012 30(12):1288-95; Budinska, E. et al. J Pathol. 2013 231(1):63-76; Sadanandam, A. et al. Nat Med. 2013 19(5):619-25; Zhang, B. et al. Nature. 2014 Jul. 20 (in press)). In an unsupervised analysis, a “PC1 signature” (PC1) was generated (Tables 2A and 2B) by selecting a list of top-ranked genes bearing positive and negative correlation with the first principal component of 326 CRC tumors. Of many signatures tested, an “EMT signature” (Tables 1A and 1B), derived from a gene expression analysis of 93 lung cancer cell lines sorted (based on their expression of CDH1 or VIM) into epithelial or mesenchymal groups, showed a very strong correlation (Pearson R=0.92, P<10⁻¹³⁵) with PC1 (Loboda, A. et al. BMC Med Genomics. 2011 4:9). This colon PC1 and lung EMT association was verified in 38 CRC cell lines and by assessment of other known EMT-related genes and microRNAs in CRC tumors (Loboda, A. et al. BMC Med Genomics. 2011 4:9).

To further assess the respective prognostic values of PC1 and EMT scores, outcomes were evaluated on a new set of 468 CRC tumors (Moffitt468) including all stages (1-4) as well as metastatic lesions, and found that both PC1 and EMT were predictive of overall survival (OS), albeit to different degrees (Table 4).

TABLE 4 Univariable models on EMT, PC1 and ΔPC1.EMT scores 95% CI 95% CI Variable Hazard Ratio lower limit Upper limit p value EMT score 1.13 0.96 1.34 0.1438 PC1 score 1.40 1.18 1.66 0.0001 ΔPC1.EMT score 1.82 1.51 2.18 <.0001

While it was clear that PC1 and EMT were highly correlated (Pearson R=0.90, P<0.0001), tumors from metastatic patients (“d_meta”) appeared to cluster, to some degree, more so towards PC1 rather than EMT (FIG. 1A, indicated by two separate regression lines for tumors with and without d_meta). This suggested that the PC1 score might contribute to better identify patients with metastatic disease or metastatic potential. Interestingly in bivariate survival models, the coefficients for PC1 and EMT were both highly significant—but of opposite sign (Table 5)—reinforcing the graphical observation showing their differential propensities for metastasis (indicating poor survival). This suggested that a signature combining both scores could predict survival better than either alone.

TABLE 5 OS Multivariable Model: PC1 and EMT scores Analysis of Maximum Likelihood Estimates Parameter Standard Chi- Hazard 95% Hazard Ratio Parameter DF Estimate Error Square Pr > ChiSq Ratio Confidence Limits EMT score 1 −1.02847 0.20329 25.5941 <.0001 0.358 0.240 0.533 PC1 score 1 1.32160 0.20542 41.3931 <.0001 3.749 2.507 5.608

To better understand the relationship of the two scores, the EMT score was subtracted from the PC1 score to produce a “difference” score (ΔPC1.EMT) (sec Tables 3A and 3B for overlapping and non-overlapping genes). As shown in FIGS. 1B and 1C, ΔPC1.EMT clearly outperformed not only EMT, but also PC1, in predicting metastasis. Correlation analysis shows that ΔPC1.EMT score had a good association with EMT (Pearson R=0.38, P<0.0001), but displayed even a stronger correlation with PC1 (Pearson R=0.74, P<0.0001), suggesting that PC1 might also include a “non-EMT” biological component. While EMT is a dominant differential molecular program in CRC (Loboda, A. et al. BMC Med Genomics. 2011 4:9), ΔPC1.EMT appeared to capture predominantly non-EMT contributions to predict metastasis (FIG. 1C, highlighted by box). Moreover, it was clear that PC1, and especially ΔPC1.EMT, outperformed EMT in progressively deciphering the degree of tumor progression of primary CRCs (stages 1 vs. 2 vs. 3 vs. 4) vs. metastatic lesions (FIG. 1D), further supporting the non-EMT bias of ΔPC1.EMT. Metastatic tumors may represent homogeneous clonal expansions of subpopulations of non-EMT(epithelial) tumor cells (Greaves, M. et al. Clonal evolution in cancer. Nature 2012 481:306-313) which might explain their higher ΔPC1.EMT score. Higher ΔPC1.EMT scores were also correlated with a higher percentage of deaths in stages 1-3 tumors (Table 6), suggesting clinical utility in predicting the use of adjunct therapy. Kaplan Meier survival analysis of quartile scores shows that a higher ΔPC1.EMT predicted poorer OS for all 468 patients (Logrank P=0.0004), and for 61 MSI patients (P=0.085) as well as 407 MSS patients (P<0.0001), respectively (FIG. 2). This result was confirmed by a Cox regression analysis in an expanded Moffitt dataset with 1988 CRC (Moffitt1988) patients (representing an additional 1520 independent cases) with both primary and metastatic tumor-derived profiles, showing that ΔPC1.EMT robustly predicted worse overall survival (beta (Cox)=2.35, i.e. HR=10.5, P=3.0×10⁻²²). Collectively, these data suggested that ΔPC1.EMT appeared to be measuring EMT as well as non-EMT biological programs that together have significant prognostic value.

TABLE 6 ΔPC1.EMT predicts deaths in Stages 1-3 CRC Stage Q1 Q2 Q3 Q4 Avg. Deaths/N Deaths/N 1 9 9 17 17 10 6/58 4/42 (10%) 2 14 21 32 36 24 29/123 14/82 (17%) 3 20 34 31 55 34 39/116 26/80 (32%)

These findings proved to be extremely robust when ΔPC1.EMT was further tested in five additional independent datasets (n=2153 CRC tumors) (Table 7) using both univariate and multivariate Cox Proportional Hazard Regression models.

TABLE 7 List of datasets used for the validation N of RNA- Follow-up Dataset Accession sample Stage Treatment Platform material Information PETACC-3 E-MTAB-990 752 2, 3 5-FU/FA and Almac FFPE OS, RFS, FOLFIRI SAR ALMAC E-MTAB-863, 359 2 Untreated^(a) Almac FFPE OS, RFS E-MTAB-864 LNCC E-GEOD- 566 1, 4 Treated- HGU133plus2 Frozen RFS 39582 Untreated GEO41258 E-GEOD- 186 1, 4 Unknown HGU133a Frozen OS, RFS 41258 GSE14333 GSE14333 290 A-D Unknown^(b) HGU133plus2 Frozen RFS ^(a)no preoperative or postoperative cancer therapy within 1 year of surgery (although therapy given after recurrence was acceptable) ^(b)standard adjuvant chemotherapy (either single agent 5-uouracil/capecitabine or 5-uouracil and oxaliplatin) or postoperative concurrent chemoradiotherapy (50.4 Gy in 28 fractions with concurrent 5-uorouracil) * related references were given in “methods”

Overall, while EMT, PC1 and ΔPC1.EMT all had hazard ratios>1.0 in univariate models, and PC1 performed better than EMT, ΔPC1.EMT consistently outperformed both in predicting OS and relapse free survival (RFS) (FIGS. 4A to 4F, and Tables 8 and 9). Moreover, the independent prognostic values of PC1 and ΔPC1.EMT were confirmed when analyzed together in a multivariate model, including other clinopathological variables (Tables 10 and 11).

TABLE 8 Univariable Cox Proportional Hazard Regression models for Overall Survival (OS), Relapse Free Survival (RFS) and Survival After Relapse (SAR) by ΔPC1.EMT score on PETACC dataset Covariates HR 1.95 u.95 p n Univariate model for OS in Stage 2 and 3 patients ΔPC1.EMT 1.56 1.32 1.84 1.16e−07 752 Univariate model for OS in Stage 3 patients ΔPC1.EMT 1.69 1.42 2.03 8.22e−09 644 Univariate model for RFS in Stage 2 and 3 patients ΔPC1.EMT 1.47 1.28 1.69 8.98e−08 752 Univariate model for RFS in Stage 3 patients ΔPC1.EMT 1.55 1.33 1.81 3.99e−08 644 Univariate model for SAR in Stage 2 and 3 patients ΔPC1.EMT 1.20 1.02 1.42 3.11e−02 291 Univariate model for SAR in Stage 3 patients ΔPC1.EMT 1.26 1.04 1.51 1.54e−02 241

TABLE 9 Univariable Cox Proportional Hazard Regression models for Overall Survival (OS) and Relapse Free Survival (RFS) - Univariate model for OS in Stage 2 and 3 - PETACC dataset Covariates HR (95% CI) p n ΔPC1.EMT 1.56 (1.32-1.84) 1.16e−07 752 PC1 1.35 (1.15-1.57) 1.54e−04 752 EMT 1.24 (1.05-1.47) 1.28e−02 752 MSI (MSS vs. MSI-H) 1.98 (1.10-3.55) 2.22e−02 752 BRAF (wt vs. mut) 0.57 (0.36-0.91) 1.89e−02 752 site (right vs. left) 1.26 (0.96-1.65) 1.03e−01 752 T stage (T12 vs. T3) 0.37 (0.16-0.83) 1.60e−02 752 T stage (T4 vs. T3) 2.00 (1.47-2.72) 9.69e−06 752 N stage (N2 vs. N1) 2.31 (1.73-3.09) 1.85e−08 752 N stage (N0 vs. N1) 1.42 (0.94-2.13) 9.63e−02 752 stage (3 vs. 2) 0.99 (0.68-1.45)  9.7e−01 752 grade (G-34 vs. G-12) 1.83 (1.24-2.72) 2.43e−03 752 SMAD4 (Any Loss vs. No Loss) 1.56 (1.15-2.11) 3.88e−03 752 BRAF.score 1.26 (1.13-1.39)  1.3e−05 752 Age 1.11 (0.97-1.26) 1.24e−01 752 LN 0.79 (0.67-0.93) 3.74e−03 750

TABLE 10 Multivariable models for Overall Survival (OS) and Relapse Free Survival (RFS) including PC1 and EMT scores - Multivariable Cox Proportional Hazard Regression model for OS in Stage2 and 3 - PETACC dataset Covariates HR 1.95 u.95 p n PC1 2.62 1.53 4.46 4.08E−004 750 EMT 0.44 0.25 0.78 4.83E−003 750 Age 1.11 0.96 1.27 1.53E−001 750 tstage (T12 vs T3) 0.47 0.2 1.08 7.42E−002 750 tstage (T4 vs T3) 2.04 1.49 2.78 8.13E−006 750 nstage (N2 vs N1) 2.13 1.58 2.87 7.20E−007 750 nstage (N0 vs N1) 1.45 0.95 2.2 8.35E−002 750 LN 0.72 0.61 0.86 2.54E−004 750 site (right vs left) 1.6 1.19 2.15 2.07E−003 750 MSI (MSS vs MSI-H) 2.48 1.33 4.63 4.19E−003 750 BRAF (wt vs mut) 0.74 0.45 1.22 2.37E−001 750

TABLE 11 Multivariable models for Overall Survival (OS) and Relapse Free Survival (RFS) including PC1.EMT score - Multivariable Cox Proportional Hazard Regression model for OS in Stage2 and 3 - PETACC dataset Covariates HR 1.95 u.95 p n ΔPC1.EMT 1.40 1.18 1.66 9.72e−05 750 Age 1.10 0.96 1.26 1.71e−01 750 tstage (T12 vs T3) 0.46 0.20 1.04 6.27e−02 750 tstage (T4 vs T3) 2.04 1.49 2.79 7.63e−06 750 nstage (N2 vs N1) 2.12 1.58 2.86 7.69e−07 750 nstage (N0 vs N1) 1.44 0.95 2.19 8.64e−02 750 LN 0.72 0.61 0.86 2.33e−04 750 site (right vs left) 1.60 1.19 2.16 1.79e−03 750 MSI (MSS vs MSI-H) 2.47 1.32 4.60 4.47e−03 750 BRAF (wt vs mut) 0.74 0.45 1.23 2.48e−01 750

To explore the molecular basis for the observed prognostic improvement of ΔPC1.EMT from its parent PC1 and EMT scores, quartile trends of these three scores vs. the number of tumors harboring observed mutations of several driver genes were examined in the Moffitt468 dataset. The ΔPC1.EMT remarkably improved the trends (relative to PC1 and EMT) to identify better prognosis tumors harboring APC mutations and worse prognosis tumors harboring BRAF (V600E) mutations, as well as tumors identified as MSI-H or Stage 4 (FIGS. 3A to 3D). This suggested the potential for ΔPC1.EMT to refine the prediction of metastasis. Moreover, it was observed that in some subgroups of combined mutations (KRAS & TP53 or BRAF & TPS3), as well as in MSI-H and Stage 1 cases, ΔPC1.EMT and EMT trended in opposite directions for distant metastasis rate (FIGS. 3E to 3H), supporting the hypothesis that ΔPC1.EMT might be measuring non-EMT components of metastasis. These data also suggested a previously dismissed role for driver genes in predicting distant metastatic potential (Vanharanta, S. et al. Cancer Cell. 2013 24(4):410-21).

The improved survival prediction with ΔPC1.EMT through capture of non-EMT components is also consistent with the hypothesis that both EMT (mesenchymal) as well as non-EMT (epithelial) cellular phenotypes must cooperate to produce metastasis (Tsuji, T et al. Cancer Res. 2009 69(18):7135-9). Tsuji et al. found that primary tumors were heterogeneous and contained both cell types (with mesenchymal cells populating the invasive front), but metastatic tumors contained only the cells originating from the epithelial type (Tsuji, T et al. Cancer Res. 2009 69(18):7135-9). Recently, new evidence has suggested a critical role for non-EMT “epithelial-like” cells in the multi-step process of metastasis (Tsuji, T et al. Cancer Res. 2008 68(24):10377-86; Giancotti, F. G. Cell. 2013 155(4):750-64; Oskarsson, T., Cell Stem Cell. 2014 14(3):306-21). For instance, cohesive epithelial migration was often observed as the predominant pattern in CRC (Chui, M. H. Int J Cancer. 2013 132(7):1487-95).

To better understand the molecular underpinnings of ΔPC1.EMT, gene expression clustering analysis was performed on the five datasets (FIG. 5A to 5B). These data show areas of strong overlap in gene expression accounting for the high correlation of EMT with PC1 (FIGS. 19A to 19E), but also show isolated, non-overlapping genes. Since the contributions of VIM (a mesenchymal gene used to create the EMT signature) and other overlapped genes were effectively diminished in ΔPC1.EMT, ΔPC1.EMT might better measure the non-EMT (epithelial) components of CRC. In addition, an analysis of the GO Process of those non-overlapping genes indicates that roughly half of the pathways were related to cell adhesion and cellular remodeling (e.g. three EMT-related pathways centered around SLUG1 were altered) (Tables 12A, 12B, 12C). Respective weighted contributions of individual signature genes of PC1 and EMT were further analyzed on the five datasets to identify the genes whose contributions changed the most from PC1 or EMT to ΔPC1.EMT (FIG. 3I). ΔPC1.EMT was represented by more epithelial and less mesenchymal gene contributions. For instance, the contribution of the epithelial marker CDH2 increased in ΔPC1.EMT, whereas the mesenchymal marker VIM and/or other EMT genes including SPARC, TCF4, COL1A2 and COL3A6 decreased.

TABLE 12A EMT GO Processes In Network Objects from Processes Total p-value FDR Data Active Data cell adhesion 988 3.917E−23 1.389E−19 66 ATR/TEM8, P-cadherin, LAMA4, FLRT2, p38 MAPK, Syk, ITGB6, EDIL3, HAS, NEPH1, COL6A1, Nectin-4, Collagen V, Calgranulin A, OSF-2, Calprotectin (S100A8/A9) complex, Galectin-1, CCL2, SRPX, CD24, Galpha(q)-specific peptide GPCRs, N-cadherin, Desmocollin 3, Caspr2, Annexin IX, R-cadherin, LAMA3 (Epiligrin), E-cadherin, FAT4, Claudin-7, TGF-beta 2, Collagen XII, LAMC2, EPDR1, Laminin 5, SHPS-1, Plakophilin 3, Nidogen, RHG7, SUSD5, BVES, CTGF, UFO, EPB41L5, MyHC, Claudin-4, Calgranulin B, PKC, Claudin-11, Tcf(Lef), IGFBP7, TGF-beta, Neurotractin, Cadherin 11, M-cadherin, COL5A1, LAMB3, WNT, DSC2, ADAM23, NPNT, Collagen III, Collagen IV, IGFBP7/8, Desmoplakin, JAM3 biological 995 5.770E−23 1.389E−19 66 ATR/TEM8, P-cadherin, LAMA4, FLRT2, adhesion p38 MAPK, Syk, ITGB6, EDIL3, HAS, NEPH1, COL6A1, Nectin-4, Collagen V, Calgranulin A, OSF-2, Calprotectin (S100A8/A9) complex, Galectin-1, CCL2, SRPX, CD24, Galpha(q)-specific peptide GPCRs, N-cadherin, Desmocollin 3, Caspr2, Annexin IX, R-cadherin, LAMA3 (Epiligrin), E-cadherin, FAT4, Claudin-7, TGF-beta 2, Collagen XII, LAMC2, EPDR1, Laminin 5, SHPS-1, Plakophilin 3, Nidogen, RHG7, SUSD5, BVES, CTGF, UFO, EPB41L5, MyHC, Claudin-4, Calgranulin B, PKC, Claudin-11, Tcf(Lef), IGFBP7, TGF-beta, Neurotractin, Cadherin 11, M-cadherin, COL5A1, LAMB3, WNT, DSC2, ADAM23, NPNT, Collagen III, Collagen IV, IGFBP7/8, Desmoplakin, JAM3 extracellular 413 2.021E−22 2.676E−19 43 RECK, FGF2, LEKTI, COL5A2, LAMA4, matrix ITGB6, HAS, P3H2, PAI1, COL6A1, organization Collagen V, OSF-2, COL4A1, GLT25D2, LAMA3 (Epiligrin), TGF-beta 2, Fibrillin 1, Collagen XII, COL1A2, LAMC2, Maspin, Tissue kallikreins, Gremlin, Laminin 5, Nidogen, Osteonectin, ELF3, CTGF, MyHC, Lysyl oxidase, PKC, TGF-beta, HAS2, Fibrillin, COL5A1, MMP-2, LAMB3, WNT, NPNT, Collagen III, Collagen IV, HAI-1, IGFBP7/8 extracellular 414 2.223E−22 2.676E−19 43 RECK, FGF2, LEKTI, COL5A2, LAMA4, structure ITGB6, HAS, P3H2, PAI1, COL6A1, organization Collagen V, OSF-2, COL4A1, GLT25D2, LAMA3 (Epiligrin), TGF-beta 2, Fibrillin 1, Collagen XII, COL1A2, LAMC2, Maspin, Tissue kallikreins, Gremlin, Laminin 5, Nidogen, Osteonectin, ELF3, CTGF, MyHC, Lysyl oxidase, PKC, TGF-beta, HAS2, Fibrillin, COL5A1, MMP-2, LAMB3, WNT, NPNT, Collagen III, Collagen IV, HAI-1, IGFBP7/8 system 4665 1.030E−19 9.920E−17 150 ELF5, RECK, Tubby, FGF2, LEKTI, development COL5A2, LAMA4, FLRT2, SPRR1A, p38 MAPK, Syk, N-chimaerin, IRF6, MR-GEF, CARP, Cx30, Keratin 19, BDNF, TTLL7, Plastin, HAS, Ankyrin-G, HEG1, Beta-fodrin, PAI1, COL6A1, PLA2, Collagen V, OSF-2, G-protein beta, Basonuclin-2, FAM101B, COL4A1, Sciellin, DAB2, CDH1, MAP7(EMAP115), FOG2, Galectin-1, Keratin 16, Pitx3, CCL2, Olfactory receptor, FHL1 (SLIM1), PGAR, CD24, Galpha(q)-specific peptide GPCRs, C1s, Zac1, UCHL1, FGF5, N- cadherin, TAJ(TNFRSF19), Caspr2, Prostasin, DYNLL, PDE, R-cadherin, LAMA3 (Epiligrin), E-cadherin, DKK3, TGF-beta 2, Fibrillin 1, Collagen XII, COL1A2, SPRR1B, GCNT3, SPRR3, LAMC2, SPTBN(spectrin1- 4), PLA2G10, DSPP, MAP-1B, Maspin, PPL(periplakin), Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, ZCCHC24, Laminin 5, G-protein beta/gamma, Myoglobin, PMP22, Nidogen, RHG7, FGFR1, Neuregulin 1, Osteonectin, Vimentin, BVES, ELF3, CTGF, ADAM-TS1, Serglycin, UFO, EPB41L5, MyHC, Lysyl oxidase, PKC, Aquaporin 3, Claudin-11, Tcf(Lef), BLNK, CRMP4, SIP1 (ZFHX1B), IGFBP7, G-protein gamma, Ankyrin-B, TCF8, TUB, TGF-beta, HAS2, FA2H, Neurotractin, Kallikrein 5, Fibrillin, Cadherin 11, CCL13, DFNA5, PKC- mu, COL5A1, Kallikrein 8, GRHL2, Necdin, CAP2, MMP-2, LAMB3, MYH14, WNT5B, Galpha(t)-specific GPCRs, Keratin 5, Kallikrein 3 (PSA), ITF2, POU2F3, WNT, FGF13, ADAM23, NPNT, Kallikrein 6 (Neurosin), Collagen III, FNBP1, Collagen IV, HAI-1, ErbB3, IGFBP7/8, M-Ras, S100B, Desmoplakin, JAM3 developmental 5978 4.175E−19 3.350E−16 174 ELF5, ESE3, RECK, Tubby, FGF2, LEKTI, process COL5A2, ATR/TEM8, LAMA4, FLRT2, SPRR1A, p38 MAPK, Syk, N-chimaerin, IRF6, MR-GEF, ITGB6, CARP, Cx30, Keratin 19, BDNF, TTLL7, Plastin, EDIL3, HAS, Ankyrin-G, Angiopoietin-like 2, HEG1, Beta-fodrin, MBNL3, PAI1, COL6A1, PLA2, Collagen V, OSF-2, G-protein beta, Basonuclin-2, PRR15, FALP, FAM101B, COL4A1, Sciellin, DAB2, CDH1, MAP7(EMAP115), FOG2, Galectin-1, PAPP- A, Keratin 16, Pitx3, CCL2, Olfactory receptor, FHL1 (SLIM1), PGAR, DMKN, PAR1, CD24, Galpha(q)-specific peptide GPCRs, C1s, Zac1, UCHL1, FGF5, N- cadherin, TAJ(TNFRSF19), Desmocollin 3, Caspr2, Prostasin, DYNLL, PDE, R-cadherin, LAMA3 (Epiligrin), E-cadherin, DKK3, TGF- beta 2, HOOK1, Fibrillin 1, Collagen XII, COL1A2, SPRR1B, GCNT3, SPRR3, LAMC2, SPTBN(spectrin1-4), PLA2G10, DSPP, STEAP4, MAP-1B, Chk2, Maspin, PPL(periplakin), Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, ZCCHC24, DLC1 (Dynein LC8a), Laminin 5, G-protein beta/gamma, Myoglobin, PMP22, Nidogen, RHG7, FGFR1, BICC1, LyGDI, Neuregulin 1, AP-1 mu subunits, TFCP2L2, Osteonectin, Vimentin, BVES, ELF3, CTGF, ADAM-TS1, Serglycin, UFO, EPB41L5, MyHC, Lysyl oxidase, PKC, Aquaporin 3, Claudin-11, Tcf(Lef), BLNK, CRMP4, SIP1 (ZFHX1B), IGFBP7, G-protein gamma, Ankyrin-B, LAF4, TCF8, TUB, TGF-beta, HAS2, FA2H, Neurotractin, Kallikrein 5, Fibrillin, Cadherin 11, CCL13, DFNA5, PKC-mu, M-cadherin, CLIP3, COL5A1, Kallikrein 8, GRHL2, Necdin, CAP2, MMP-2, LAMB3, MYH14, WNT5B, Galpha(t)-specific GPCRs, Keratin 5, Kallikrein 3 (PSA), ITF2, POU2F3, WNT, FGF13, ADAM23, NPNT, Kallikrein 6 (Neurosin), Collagen III, FNBP1, RBM24, Collagen IV, HAI-1, ErbB3, IGFBP7/8, M- Ras, S100B, Desmoplakin, JAM3 multicellular 5406 1.414E−18 9.694E−16 162 ELF5, ESE3, RECK, Tubby, FGF2, LEKTI, organismal COL5A2, LAMA4, FLRT2, SPRR1A, p38 development MAPK, Syk, N-chimaerin, IRF6, MR-GEF, ITGB6, CARP, Cx30, Keratin 19, BDNF, TTLL7, Plastin, EDIL3, HAS, Ankyrin-G, Angiopoietin-like 2, HEG1, Beta-fodrin, MBNL3, PAI1, COL6A1, PLA2, Collagen V, OSF-2, G-protein beta, Basonuclin-2, PRR15, FAM101B, COL4A1, Sciellin, DAB2, CDH1, MAP7(EMAP115), FOG2, Galectin-1, Keratin 16, Pitx3, CCL2, Olfactory receptor, FHL1 (SLIM1), PGAR, CD24, Galpha(q)-specific peptide GPCRs, C1s, Zac1, UCHL1, FGF5, N- cadherin, TAJ(TNFRSF19), Desmocollin 3, Caspr2, Prostasin, DYNLL, PDE, R-cadherin, LAMA3 (Epiligrin), E-cadherin, DKK3, TGF- beta 2, HOOK1, Fibrillin 1, Collagen XII, COL1A2, SPRR1B, GCNT3, SPRR3, LAMC2, SPTBN(spectrin1-4), PLA2G10, DSPP, MAP-1B, Maspin, PPL(periplakin), Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, ZCCHC24, Laminin 5, G-protein beta/gamma, Myoglobin, PMP22, Nidogen, RHG7, FGFR1, BICC1, LyGDI, Neuregulin 1, TFCP2L2, Osteonectin, Vimentin, BVES, ELF3, CTGF, ADAM-TS1, Serglycin, UFO, EPB41L5, MyHC, Lysyl oxidase, PKC, Aquaporin 3, Claudin-11, Tcf(Lef), BLNK, CRMP4, SIP1 (ZFHX1B), IGFBP7, G-protein gamma, Ankyrin-B, LAF4, TCF8, TUB, TGF- beta, HAS2, FA2H, Neurotractin, Kallikrein 5, Fibrillin, Cadherin 11, CCL13, DFNA5, PKC- mu, COL5A1, Kallikrein 8, GRHL2, Necdin, CAP2, MMP-2, LAMB3, MYH14, WNT5B, Galpha(t)-specific GPCRs, Keratin 5, Kallikrein 3 (PSA), ITF2, POU2F3, WNT, FGF13, ADAM23, NPNT, Kallikrein 6 (Neurosin), Collagen III, FNBP1, Collagen IV, HAI-1, ErbB3, IGFBP7/8, M-Ras, S100B, Desmoplakin, JAM3 anatomical 2492 1.611E−18 9.694E−16 100 Tubby, FGF2, COL5A2, ATR/TEM8, FLRT2, structure p38 MAPK, Syk, N-chimaerin, CARP, Cx30, morphogenesis Keratin 19, BDNF, HAS, Ankyrin-G, HEG1, Beta-fodrin, PAI1, COL6A1, PLA2, Collagen V, Basonuclin-2, FAM101B, COL4A1, DAB2, MAP7(EMAP115), FOG2, Pitx3, CCL2, Olfactory receptor, FHL1 (SLIM1), PGAR, PAR1, CD24, Galpha(q)-specific peptide GPCRs, UCHL1, N-cadherin, DYNLL, PDE, R-cadherin, E-cadherin, DKK3, TGF-beta 2, COL1A2, GCNT3, SPTBN(spectrin1-4), PLA2G10, DSPP, MAP- 1B, Maspin, Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, DLC1 (Dynein LC8a), G-protein beta/gamma, PMP22, RHG7, FGFR1, Neuregulin 1, BVES, ELF3, CTGF, ADAM-TS1, UFO, EPB41L5, MyHC, PKC, Aquaporin 3, Tcf(Lef), CRMP4, SIP1 (ZFHX1B), G-protein gamma, Ankyrin-B, LAF4, TCF8, TGF-beta, HAS2, Fibrillin, Cadherin 11, CCL13, PKC-mu, COL5A1, Kallikrein 8, GRHL2, Necdin, CAP2, MMP-2, MYH14, WNT5B, Kallikrein 3 (PSA), WNT, NPNT, Collagen III, Collagen IV, HAI-1, ErbB3, IGFBP7/8, S100B, Desmoplakin, JAM3 anatomical 5314 1.296E−17 6.934E−15 158 ELF5, ESE3, RECK, Tubby, FGF2, LEKTI, structure COL5A2, ATR/TEM8, LAMA4, FLRT2, development SPRR1A, p38 MAPK, Syk, N-chimaerin, IRF6, MR-GEF, CARP, Cx30, Keratin 19, BDNF, TTLL7, Plastin, HAS, Ankyrin-G, HEG1, Beta-fodrin, PAI1, COL6A1, PLA2, Collagen V, OSF-2, G-protein beta, Basonuclin-2, FAM101B, COL4A1, Sciellin, DAB2, CDH1, MAP7(EMAP115), FOG2, Galectin-1, Keratin 16, Pitx3, CCL2, Olfactory receptor, FHL1 (SLIM1), PGAR, PAR1, CD24, Galpha(q)-specific peptide GPCRs, C1s, Zac1, UCHL1, FGF5, N-cadherin, TAJ(TNFRSF19), Desmocollin 3, Caspr2, Prostasin, DYNLL, PDE, R-cadherin, LAMA3 (Epiligrin), E-cadherin, DKK3, TGF-beta 2, HOOK1, Fibrillin 1, Collagen XII, COL1A2, SPRR1B, GCNT3, SPRR3, LAMC2, SPTBN(spectrin1-4), PLA2G10, DSPP, MAP- 1B, Maspin, PPL(periplakin), Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, ZCCHC24, DLC1 (Dynein LC8a), Laminin 5, G-protein beta/gamma, Myoglobin, PMP22, Nidogen, RHG7, FGFR1, Neuregulin 1, Osteonectin, Vimentin, BVES, ELF3, CTGF, ADAM-TS1, Serglycin, UFO, EPB41L5, MyHC, Lysyl oxidase, PKC, Aquaporin 3, Claudin-11, Tcf(Lef), BLNK, CRMP4, SIP1 (ZFHX1B), IGFBP7, G-protein gamma, Ankyrin-B, LAF4, TCF8, TUB, TGF- beta, HAS2, FA2H, Neurotractin, Kallikrein 5, Fibrillin, Cadherin 11, CCL13, DFNA5, PKC- mu, M-cadherin, COL5A1, Kallikrein 8, GRHL2, Necdin, CAP2, MMP-2, LAMB3, MYH14, WNT5B, Galpha(t)-specific GPCRs, Keratin 5, Kallikrein 3 (PSA), ITF2, POU2F3, WNT, FGF13, ADAM23, NPNT, Kallikrein 6 (Neurosin), Collagen III, FNBP1, Collagen IV, HAI-1, ErbB3, IGFBP7/8, M-Ras, S100B, Desmoplakin, JAM3 cell 3602 1.740E−17 8.378E−15 123 ELF5, ESE3, Tubby, FGF2, LEKTI, COL5A2, differentiation ATR/TEM8, LAMA4, FLRT2, SPRR1A, p38 MAPK, Syk, N-chimaerin, IRF6, CARP, Keratin 19, BDNF, TTLL7, HAS, Ankyrin-G, HEG1, Beta-fodrin, COL6A1, PLA2, Collagen V, FALP, FAM101B, COL4A1, Sciellin, DAB2, CDH1, MAP7(EMAP115), FOG2, Galectin-1, PAPP-A, Pitx3, CCL2, Olfactory receptor, FHL1 (SLIM1), PGAR, DMKN, CD24, Galpha(q)-specific peptide GPCRs, C1s, Zac1, UCHL1, FGF5, N-cadherin, Caspr2, DYNLL, PDE, R-cadherin, E- cadherin, TGF-beta 2, HOOK1, SPRR1B, SPRR3, SPTBN(spectrin1-4), PLA2G10, DSPP, STEAP4, MAP-1B, PPL(periplakin), Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, ZCCHC24, Laminin 5, G-protein beta/gamma, Myoglobin, PMP22, FGFR1, Neuregulin 1, AP-1 mu subunits, Vimentin, BVES, ELF3, CTGF, UFO, EPB41L5, MyHC, PKC, Tcf(Lef), BLNK, CRMP4, SIP1 (ZFHX1B), G-protein gamma, Ankyrin-B, TCF8, TGF-beta, HAS2, FA2H, Neurotractin, Cadherin 11, CCL13, DFNA5, PKC-mu, M- cadherin, CLIP3, COL5A1, Kallikrein 8, Necdin, CAP2, MMP-2, LAMB3, MYH14, WNT5B, ITF2, POU2F3, WNT, FGF13, NPNT, Kallikrein 6 (Neurosin), Collagen III, FNBP1, RBM24, Collagen IV, ErbB3, IGFBP7/8, S100B, Desmoplakin, JAM3 tissue 1879 4.429E−17 1.938E−14 82 ELF5, ESE3, Tubby, FGF2, LEKTI, SPRR1A, development p38 MAPK, IRF6, CARP, HAS, HEG1, PAI1, PLA2, Collagen V, OSF-2, Basonuclin-2, FAM101B, COL4A1, Sciellin, DAB2, CDH1, MAP7(EMAP115), FOG2, Keratin 16, Pitx3, CD24, Galpha(q)-specific peptide GPCRs, Zac1, TAJ(TNFRSF19), Prostasin, PDE, LAMA3 (Epiligrin), E-cadherin, TGF-beta 2, HOOK1, SPRR1B, GCNT3, SPRR3, LAMC2, DSPP, Maspin, PPL(periplakin), Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, Laminin 5, RHG7, FGFR1, Neuregulin 1, Vimentin, ELF3, CTGF, Serglycin, UFO, EPB41L5, MyHC, PKC, Tcf(Lef), SIP1 (ZFHX1B), TCF8, TGF-beta, HAS2, FA2H, Kallikrein 5, COL5A1, GRHL2, MMP-2, LAMB3, WNT5B, Keratin 5, Kallikrein 3 (PSA), POU2F3, WNT, NPNT, Kallikrein 6 (Neurosin), Collagen IV, HAI-1, ErbB3, IGFBP7/8, Desmoplakin, JAM3 single-organism 13941 1.487E−16 5.968E−14 290 ELF5, ESE3, AKAP12, RECK, Tubby, FGF2, process IL13RA2, LEKTI, COL5A2, PNMA2, ATR/TEM8, P-cadherin, BCMP84, LAMA4, CDS1, FLRT2, SPRR1A, INPP4B, p38 MAPK, Syk, Galpha(i)-specific prostanoid GPCRs, ATP8B2, N-chimaerin, TBXA2R, IRF6, Tubulin alpha 1A, MR-GEF, ITGB6, CARP, Cx30, Keratin 19, UBAP2L, FUT3, Calgizzarin, BDNF, TTLL7, Plastin, EDIL3, HAS, PTGIS, Ankyrin-G, NEPH1, Cingulin, Angiopoietin-like 2, P3H2, HEG1, Beta- fodrin, MBNL3, PAI1, COL6A1, Nectin-4, STX19, PLA2, Collagen V, MATE1, Calgranulin A, OSF-2, PPP1R14C, GPX2, GLUT3, EMP3, Synaptotagmin VII, G-protein beta, Basonuclin-2, PRR15, GPR87, DIO2, FALP, FAM101B, TMSL8, CPA4, FGD2, COL4A1, TMC4, Matriptase, MARVELD3, Calprotectin (S100A8/A9) complex, Sciellin, CYBRD1, CNK1, BSPRY, DAB2, CDH1, MAP7(EMAP115), FOG2, Galectin-1, PAPP- A, Keratin 16, Pitx3, GLT25D2, SYDE1, CCL2, SRPX, Olfactory receptor, FHL1 (SLIM1), PGAR, Galpha(q)-specific prostanoid GPCRs, KIAA1043, AKS, NAP1L3, DMKN, LIPE, Annexin VI, PAR1, MAPBPIP, CD24, Galpha(q)-specific peptide GPCRs, C1s, Zac1, UCHL1, FGF5, Tubulin alpha, Synaptotagmin, IL1RN, BPGAP1, CDP-diacylglycerol synthase, N-cadherin, TAJ(TNFRSF19), Desmocollin 3, Caspr2, PRR5, Prostasin, DYNLL, PDE, TOX3, Annexin IX, R-cadherin, LAMA3 (Epiligrin), E-cadherin, FAT4, DKK3, DOCK10, PRR5- ARHGAP8, Claudin-7, TGF-beta 2, HOOK1, Myosin Vb, PTX3, Fibrillin 1, Collagen XII, COL1A2, SPRR1B, B7-H4, GCNT3, SPRR3, LAMC2, Tricellulin, G-protein beta-4, SPTBN(spectrin1-4), PLA2G10, DSPP, STEAP4, MAP-1B, Chk2, Factor H, Maspin, PPL(periplakin), Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Kallikrein 1, RASGEF1B, Gremlin, NSP1, ZCCHC24, DLC1 (Dynein LC8a), EPDR1, ATP2C2, Laminin 5, SHPS-1, G-protein beta/gamma, Gpc6, Myoglobin, Plakophilin 3, PMP22, Nidogen, RHG7, FGFR1, TRPA1, BICC1, Rab-25, HBP17, LyGDI, AOX1, Neuregulin 1, AP-1 mu subunits, TFCP2L2, SUSD5, Osteonectin, Vimentin, BVES, ELF3, CTGF, ADAM-TS1, Serglycin, ATP8B3, UFO, EPB41L5, p38delta (MAPK13), MyHC, Lysyl oxidase, Claudin-4, Calgranulin B, PKC, Aquaporin 3, FR-alpha, GPR176, Claudin-11, Tcf(Lef), WaspIP, MPP7, BLNK, CRMP4, SIP1 (ZFHX1B), IGFBP7, G-protein gamma, Ankyrin-B, LAF4, PDE7B, G-protein gamma 11, TCF8, TUB, PAK6, TGF-beta, HAS2, alpha-ENaC, FA2H, ADAM28, Neurotractin, Kallikrein 5, Fibrillin, AP1S2, Cadherin 11, CCL13, DFNA5, NGAL, PKC-mu, M-cadherin, GPR110, CLIP3, COL5A1, Kallikrein 8, GRHL2, Necdin, CAP2, MMP-2, LAMB3, SLC6A14, PTAFR, FSTL1, Aggrecanase-2, AP1M2, CEACAM6, WFDC2, MYH14, WNT5B, AP-1 sigma subunits, Galpha(t)- specific GPCRs, Keratin 5, Kallikrein 3 (PSA), ITF2, RASEF, POU2F3, WNT, F16P, TMC5, DAPP1, DSC2, Kallikrein 10 (KLK10), FGF13, ADAM23, NPNT, Kallikrein 6 (Neurosin), FXYD3, Collagen III, FNBP1, RBM24, ELP70, Collagen IV, ChAF1 subunit B, HAI-1, ErbB3, HAI-2, IGFBP7/8, M-Ras, Mucin 20, S100B, Desmoplakin, S100P, JAM3 cellular 3745 3.888E−16 1.440E−13 123 ELF5, ESE3, Tubby, FGF2, LEKTI, COL5A2, developmental ATR/TEM8, LAMA4, FLRT2, SPRR1A, p38 process MAPK, Syk, N-chimaerin, IRF6, CARP, Keratin 19, BDNF, TTLL7, HAS, Ankyrin-G, HEG1, Beta-fodrin, COL6A1, PLA2, Collagen V, FALP, FAM101B, COL4A1, Sciellin, DAB2, CDH1, MAP7(EMAP115), FOG2, Galectin-1, PAPP-A, Pitx3, CCL2, Olfactory receptor, FHL1 (SLIM1), PGAR, DMKN, CD24, Galpha(q)-specific peptide GPCRs, C1s, Zac1, UCHL1, FGF5, N-cadherin, Caspr2, DYNLL, PDE, R-cadherin, E- cadherin, TGF-beta 2, HOOK1, SPRR1B, SPRR3, SPTBN(spectrin1-4), PLA2G10, DSPP, STEAP4, MAP-1B, PPL(periplakin), Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, ZCCHC24, Laminin 5, G-protein beta/gamma, Myoglobin, PMP22, FGFR1, Neuregulin 1, AP-1 mu subunits, Vimentin, BVES, ELF3, CTGF, UFO, EPB41L5, MyHC, PKC, Tcf(Lef), BLNK, CRMP4, SIP1 (ZFHX1B), G-protein gamma, Ankyrin-B, TCF8, TGF-beta, HAS2, FA2H, Neurotractin, Cadherin 11, CCL13, DFNA5, PKC-mu, M- cadherin, CLIP3, COL5A1, Kallikrein 8, Necdin, CAP2, MMP-2, LAMB3, MYH14, WNT5B, ITF2, POU2F3, WNT, FGF13, NPNT, Kallikrein 6 (Neurosin), Collagen III, FNBP1, RBM24, Collagen IV, ErbB3, IGFBP7/8, S100B, Desmoplakin, JAM3 response to 1415 1.288E−15 4.429E−13 67 FGF2, P-cadherin, p38 MAPK, Syk, wounding Galpha(i)-specific prostanoid GPCRs, TBXA2R, ITGB6, BDNF, Plastin, PAI1, PLA2, Collagen V, Calgranulin A, Synaptotagmin VII, G-protein beta, Calprotectin (S100A8/A9) complex, FOG2, Galectin-1, CCL2, Galpha(q)-specific prostanoid GPCRs, PAR1, CD24, Galpha(q)- specific peptide GPCRs, Tubulin alpha, Synaptotagmin, IL1RN, PDE, TGF-beta 2, PTX3, COL1A2, SPRR3, MAP-1B, Tissue kallikreins, SHPS-1, G-protein beta/gamma, AOX1, Neuregulin 1, Osteonectin, Vimentin, ELF3, CTGF, Serglycin, UFO, MyHC, Lysyl oxidase, Calgranulin B, PKC, BLNK, CRMP4, G-protein gamma, TGF-beta, CCL13, PKC- mu, COL5A1, Kallikrein 8, MMP-2, PTAFR, WNT5B, Kallikrein 3 (PSA), POU2F3, WNT, Kallikrein 6 (Neurosin), Collagen III, ErbB3, IGFBP7/8, Desmoplakin, JAM3 skin 406 4.949E−15 1.588E−12 34 LEKTI, COL5A2, SPRR1A, IRF6, Collagen development V, Sciellin, Keratin 16, TAJ(TNFRSF19), Prostasin, LAMA3 (Epiligrin), TGF-beta 2, COL1A2, SPRR1B, SPRR3, LAMC2, PPL(periplakin), Tissue kallikreins, TCF7L2 (TCF4), Laminin 5, FGFR1, ELF3, CTGF, Tcf(Lef), TGF-beta, FA2H, Kallikrein 5, COL5A1, LAMB3, Keratin 5, POU2F3, WNT, Collagen III, IGFBP7/8, Desmoplakin regulation of 1910 1.227E−14 3.692E−12 78 Tubby, FGF2, p38 MAPK, Syk, N-chimaerin, cellular CARP, BDNF, HAS, Ankyrin-G, NEPH1, component Beta-fodrin, PAI1, PLA2, Collagen V, organization Calgranulin A, TMSL8, FGD2, Calprotectin (S100A8/A9) complex, DAB2, Galectin-1, CCL2, FHL1 (SLIM1), CD24, Galpha(q)- specific peptide GPCRs, Synaptotagmin, N- cadherin, DYNLL, R-cadherin, TGF-beta 2, PTX3, FAM110C, SPRR1B, SPRR3, SPTBN(spectrin1-4), MAP-1B, Tissue kallikreins, TCF7L2 (TCF4), Gremlin, DLC1 (Dynein LC8a), SHPS-1, G-protein beta/gamma, PRSS11 (HtrA1), PMP22, RHG7, FGFR1, Neuregulin 1, Vimentin, BVES, CTGF, EPB41L5, MyHC, Nelin, Calgranulin B, PKC, Tcf(Lef), MPP7, CRMP4, IGFBP7, G-protein gamma, TUB, TGF-beta, Neurotractin, CCL13, NGAL, PKC-mu, CLIP3, COL5A1, Kallikrein 8, Necdin, MYH14, WNT, F16P, FGF13, SH3YL1, Kallikrein 6 (Neurosin), IGFBP7/8, S100B, JAM3 cell 792 1.361E−14 3.854E−12 47 COL5A2, ATR/TEM8, FLRT2, N-chimaerin, morphogenesis BDNF, HAS, Ankyrin-G, HEG1, Beta-fodrin, involved in COL6A1, PLA2, Collagen V, FAM101B, differentiation COL4A1, DAB2, Olfactory receptor, CD24, Galpha(q)-specific peptide GPCRs, UCHL1, R-cadherin, E-cadherin, TGF-beta 2, SPTBN(spectrin1-4), PLA2G10, MAP-1B, Gremlin, FGFR1, BVES, UFO, EPB41L5, MyHC, PKC, Tcf(Lef), CRMP4, Ankyrin-B, TGF-beta, HAS2, Cadherin 11, COL5A1, Necdin, CAP2, MYH14, WNT, Collagen III, Collagen IV, ErbB3, S100B cell-cell 486 3.259E−14 8.718E−12 36 P-cadherin, p38 MAPK, Syk, NEPH1, Nectin- adhesion 4, Calgranulin A, Calprotectin (S100A8/A9) complex, Galectin-1, CD24, N-cadherin, Desmocollin 3, Annexin IX, R-cadherin, LAMA3 (Epiligrin), E-cadherin, FAT4, Claudin-7, TGF-beta 2, Laminin 5, BVES, CTGF, MyHC, Claudin-4, Calgranulin B, Claudin-11, Tcf(Lef), TGF-beta, Neurotractin, Cadherin 11, M-cadherin, WNT, DSC2, NPNT, IGFBP7/8, Desmoplakin, JAM3 single-organism 12767 8.058E−14 2.042E−11 269 ELF5, ESE3, AKAP12, RECK, Tubby, FGF2, cellular process IL13RA2, LEKTI, COL5A2, PNMA2, ATR/TEM8, P-cadherin, BCMP84, LAMA4, CDS1, FLRT2, SPRR1A, INPP4B, p38 MAPK, Syk, Galpha(i)-specific prostanoid GPCRs, ATP8B2, N-chimaerin, TBXA2R, IRF6, Tubulin alpha 1A, MR-GEF, ITGB6, CARP, Cx30, Keratin 19, UBAP2L, FUT3, Calgizzarin, BDNF, TTLL7, Plastin, EDIL3, HAS, PTGIS, Ankyrin-G, NEPH1, Cingulin, P3H2, HEG1, Beta-fodrin, PAI1, COL6A1, Nectin-4, STX19, PLA2, Collagen V, MATE1, Calgranulin A, OSF-2, PPP1R14C, GLUT3, EMP3, G-protein beta, GPR87, DIO2, FALP, FAM101B, TMSL8, CPA4, FGD2, COL4A1, Matriptase, MARVELD3, Calprotectin (S100A8/A9) complex, Sciellin, CYBRD1, CNK1, DAB2, CDH1, MAP7(EMAP115), FOG2, Galectin-1, PAPP- A, Keratin 16, Pitx3, GLT25D2, SYDE1, CCL2, SRPX, Olfactory receptor, FHL1 (SLIM1), PGAR, Galpha(q)-specific prostanoid GPCRs, KIAA1043, AK5, NAP1L3, DMKN, PAR1, MAPBPIP, CD24, Galpha(q)-specific peptide GPCRs, C1s, Zac1, UCHL1, FGF5, Tubulin alpha, Synaptotagmin, IL1RN, BPGAP1, CDP- diacylglycerol synthase, N-cadherin, TAJ(TNFRSF19), Desmocollin 3, Caspr2, PRR5, DYNLL, PDE, TOX3, Annexin IX, R- cadherin, LAMA3 (Epiligrin), E-cadherin, FAT4, DKK3, DOCK10, PRR5-ARHGAP8, Claudin-7, TGF-beta 2, HOOK1, Myosin Vb, Fibrillin 1, Collagen XII, COL1A2, SPRR1B, B7-H4, SPRR3, LAMC2, Tricellulin, G- protein beta-4, SPTBN(spectrin1-4), PLA2G10, DSPP, STEAP4, MAP-1B, Chk2, Factor H, Maspin, PPL(periplakin), Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Kallikrein 1, RASGEF1B, Gremlin, NSP1, ZCCHC24, DLC1 (Dynein LC8a), EPDR1, ATP2C2, Laminin 5, SHPS-1, G-protein beta/gamma, Gpc6, Myoglobin, Plakophilin 3, PMP22, Nidogen, RHG7, FGFR1, TRPA1, Rab-25, HBP17, LyGDI, AOX1, Neuregulin 1, AP-1 mu subunits, SUSD5, Osteonectin, Vimentin, BVES, ELF3, CTGF, ADAM-TS1, Serglycin, ATP8B3, UFO, EPB41L5, p38delta (MAPK13), MyHC, Lysyl oxidase, Claudin-4, Calgranulin B, PKC, Aquaporin 3, FR-alpha, GPR176, Claudin-11, Tcf(Lef), WaspIP, MPP7, BLNK, CRMP4, SIP1 (ZFHX1B), IGFBP7, G-protein gamma, Ankyrin-B, PDE7B, G-protein gamma 11, TCF8, TUB, PAK6, TGF-beta, HAS2, alpha-ENaC, FA2H, ADAM28, Neurotractin, Fibrillin, AP1S2, Cadherin 11, CCL13, DFNA5, NGAL, PKC- mu, M-cadherin, GPR110, CLIP3, COL5A1, Kallikrein 8, Necdin, CAP2, MMP-2, LAMB3, SLC6A14, PTAFR, FSTL1, Aggrecanase-2, AP1M2, CEACAM6, MYH14, WNT5B, AP-1 sigma subunits, Galpha(t)-specific GPCRs, Keratin 5, Kallikrein 3 (PSA), ITF2, RASEF, POU2F3, WNT, F16P, DAPP1, DSC2, Kallikrein 10 (KLK10), FGF13, ADAM23, NPNT, Kallikrein 6 (Neurosin), FXYD3, Collagen III, FNBP1, RBM24, Collagen IV, ChAF1 subunit B, HAI-1, ErbB3, HAI-2, IGFBP7/8, M-Ras, Mucin 20, S100B, Desmoplakin, S100P, JAM3 single- 7762 1.108E−13 2.569E−11 192 ELF5, ESE3, RECK, Tubby, FGF2, LEKTI, multicellular COL5A2, P-cadherin, LAMA4, FLRT2, organism process SPRR1A, p38 MAPK, Syk, Galpha(i)-specific prostanoid GPCRs, N-chimaerin, TBXA2R, IRF6, MR-GEF, ITGB6, CARP, Cx30, Keratin 19, UBAP2L, FUT3, BDNF, TTLL7, Plastin, EDIL3, HAS, Ankyrin-G, NEPH1, Angiopoietin-like 2, P3H2, HEG1, Beta- fodrin, MBNL3, PAI1, COL6A1, PLA2, Collagen V, Calgranulin A, OSF-2, GPX2, G- protein beta, Basonuclin-2, PRR15, FAM101B, COL4A1, Calprotectin (S100A8/A9) complex, Sciellin, DAB2, CDH1, MAP7(EMAP115), FOG2, Galectin-1, Keratin 16, Pitx3, CCL2, Olfactory receptor, FHL1 (SLIM1), PGAR, Galpha(q)-specific prostanoid GPCRs, LIPE, PAR1, CD24, Galpha(q)-specific peptide GPCRs, C1s, Zac1, UCHL1, FGF5, Tubulin alpha, Synaptotagmin, IL1RN, N-cadherin, TAJ(TNFRSF19), Desmocollin 3, Caspr2, Prostasin, DYNLL, PDE, R-cadherin, LAMA3 (Epiligrin), E-cadherin, DKK3, TGF-beta 2, HOOK1, Fibrillin 1, Collagen XII, COL1A2, SPRR1B, B7-H4, GCNT3, SPRR3, LAMC2, Tricellulin, SPTBN(spectrin1-4), PLA2G10, DSPP, MAP-1B, Maspin, PPL(periplakin), Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, ZCCHC24, DLC1 (Dynein LC8a), Laminin 5, SHPS-1, G-protein beta/gamma, Myoglobin, PMP22, Nidogen, RHG7, FGFR1, TRPA1, BICC1, LyGDI, Neuregulin 1, TFCP2L2, Osteonectin, Vimentin, BVES, ELF3, CTGF, ADAM-TS1, Serglycin, ATP8B3, UFO, EPB41L5, MyHC, Lysyl oxidase, Calgranulin B, PKC, Aquaporin 3, GPR176, Claudin-11, Tcf(Lef), BLNK, CRMP4, SIP1 (ZFHX1B), IGFBP7, G-protein gamma, Ankyrin-B, LAF4, PDE7B, TCF8, TUB, TGF-beta, HAS2, alpha-ENaC, FA2H, Neurotractin, Kallikrein 5, Fibrillin, Cadherin 11, CCL13, DFNA5, PKC-mu, COL5A1, Kallikrein 8, GRHL2, Necdin, CAP2, MMP-2, LAMB3, PTAFR, MYH14, WNT5B, Galpha(t)-specific GPCRs, Keratin 5, Kallikrein 3 (PSA), ITF2, POU2F3, WNT, DSC2, FGF13, ADAM23, NPNT, Kallikrein 6 (Neurosin), Collagen III, FNBP1, ELP70, Collagen IV, HAI-1, ErbB3, IGFBP7/8, M- Ras, S100B, Desmoplakin, S100P, JAM3 multicellular 8059 1.120E−13 2.569E−11 197 ELF5, ESE3, RECK, Tubby, FGF2, LEKTI, organismal COL5A2, P-cadherin, LAMA4, FLRT2, process SPRR1A, p38 MAPK, Syk, Galpha(i)-specific prostanoid GPCRs, N-chimaerin, TBXA2R, IRF6, MR-GEF, ITGB6, CARP, Cx30, Keratin 19, UBAP2L, FUT3, Calgizzarin, BDNF, TTLL7, Plastin, EDIL3, HAS, Ankyrin-G, NEPH1, Angiopoietin-like 2, P3H2, HEG1, Beta-fodrin, MBNL3, PAI1, COL6A1, PLA2, Collagen V, Calgranulin A, OSF-2, GPX2, G-protein beta, Basonuclin-2, PRR15, FAM101B, COL4A1, Calprotectin (S100A8/A9) complex, Sciellin, DAB2, CDH1, MAP7(EMAP115), FOG2, Galectin-1, PAPP-A, Keratin 16, Pitx3, CCL2, Olfactory receptor, FHL1 (SLIM1), PGAR, Galpha(q)- specific prostanoid GPCRs, LIPE, PAR1, CD24, Galpha(q)-specific peptide GPCRs, C1s, Zac1, UCHL1, FGF5, Tubulin alpha, Synaptotagmin, IL1RN, N-cadherin, TAJ(TNFRSF19), Desmocollin 3, Caspr2, Prostasin, DYNLL, PDE, R-cadherin, LAMA3 (Epiligrin), E-cadherin, DKK3, TGF-beta 2, HOOK1, Fibrillin 1, Collagen XII, COL1A2, SPRR1B, B7-H4, GCNT3, SPRR3, LAMC2, Tricellulin, SPTBN(spectrin1-4), PLA2G10, DSPP, MAP-1B, Maspin, PPL(periplakin), Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, ZCCHC24, DLC1 (Dynein LC8a), Laminin 5, SHPS-1, G-protein beta/gamma, Myoglobin, PMP22, Nidogen, RHG7, FGFR1, TRPA1, BICC1, LyGDI, Neuregulin 1, TFCP2L2, Osteonectin, Vimentin, BVES, ELF3, CTGF, ADAM-TS1, Serglycin, ATP8B3, UFO, EPB41L5, MyHC, Lysyl oxidase, Claudin-4, Calgranulin B, PKC, Aquaporin 3, GPR176, Claudin-11, Tcf(Lef), BLNK, CRMP4, SIP1 (ZFHX1B), IGFBP7, G-protein gamma, Ankyrin-B, LAF4, PDE7B, TCF8, TUB, TGF-beta, HAS2, alpha-ENaC, FA2H, ADAM28, Neurotractin, Kallikrein 5, Fibrillin, Cadherin 11, CCL13, DFNA5, PKC- mu, COL5A1, Kallikrein 8, GRHL2, Necdin, CAP2, MMP-2, LAMB3, PTAFR, WFDC2, MYH14, WNT5B, Galpha(t)-specific GPCRs, Keratin 5, Kallikrein 3 (PSA), ITF2, POU2F3, WNT, DSC2, FGF13, ADAM23, NPNT, Kallikrein 6 (Neurosin), Collagen III, FNBP1, ELP70, Collagen IV, HAI-1, ErbB3, IGFBP7/8, M-Ras, S100B, Desmoplakin, S100P, JAM3 cellular 5002 1.297E−13 2.839E−11 142 RECK, Tubby, FGF2, LEKTI, COL5A2, component ATR/TEM8, P-cadherin, LAMA4, FLRT2, organization p38 MAPK, N-chimaerin, Tubulin alpha 1A, ITGB6, CARP, Keratin 19, BDNF, Plastin, HAS, Ankyrin-G, NEPH1, P3H2, HEG1, Beta-fodrin, PAI1, COL6A1, Nectin-4, PLA2, Collagen V, OSF-2, Synaptotagmin VII, FAM101B, TMSL8, CPA4, FGD2, COL4A1, MARVELD3, Calprotectin (S100A8/A9) complex, DAB2, CDH1, MAP7(EMAP115), Keratin 16, GLT25D2, CCL2, SRPX, Olfactory receptor, PGAR, KIAA1043, NAP1L3, LIPE, PAR1, CD24, Galpha(q)- specific peptide GPCRs, UCHL1, Tubulin alpha, Synaptotagmin, N-cadherin, Caspr2, PRR5, DYNLL, PDE, R-cadherin, LAMA3 (Epiligrin), E-cadherin, FAT4, TGF-beta 2, HOOK1, Fibrillin 1, Collagen XII, COL1A2, LAMC2, Tricellulin, SPTBN(spectrin1-4), PLA2G10, MAP-1B, Chk2, Maspin, Tissue kallikreins, TCF7L2 (TCF4), Gremlin, DLC1 (Dynein LC8a), Laminin 5, SHPS-1, PMP22, Nidogen, RHG7, FGFR1, Rab-25, LyGDI, Neuregulin 1, AP-1 mu subunits, Osteonectin, Vimentin, BVES, ELF3, CTGF, Serglycin, ATP8B3, UFO, EPB41L5, MyHC, Lysyl oxidase, Calgranulin B, PKC, FR-alpha, Tcf(Lef), WaspIP, MPP7, CRMP4, Ankyrin- B, PAK6, TGF-beta, HAS2, FA2H, Fibrillin, Cadherin 11, CCL13, NGAL, PKC-mu, M- cadherin, COL5A1, Kallikrein 8, Necdin, CAP2, MMP-2, LAMB3, MYH14, Keratin 5, ITF2, WNT, F16P, FGF13, NPNT, Collagen III, Collagen IV, ChAF1 subunit B, HAI-1, ErbB3, IGFBP7/8, M-Ras, Mucin 20, S100B, Desmoplakin cellular 1526 1.610E−13 3.371E−11 66 FGF2, COL5A2, FLRT2, p38 MAPK, Syk, N- component chimaerin, BDNF, Ankyrin-G, Beta-fodrin, movement COL6A1, PLA2, Collagen V, Calgranulin A, COL4A1, Matriptase, Calprotectin (S100A8/A9) complex, CCL2, Olfactory receptor, CD24, Galpha(q)-specific peptide GPCRs, UCHL1, N-cadherin, DYNLL, PDE, R-cadherin, TGF-beta 2, COL1A2, SPTBN(spectrin1-4), PLA2G10, MAP-1B, Maspin, OVOL2, Gremlin, DLC1 (Dynein LC8a), SHPS-1, Gpc6, FGFR1, LyGDI, Neuregulin 1, Vimentin, CTGF, UFO, EPB41L5, MyHC, Calgranulin B, PKC, Tcf(Lef), WaspIP, CRMP4, SIP1 (ZFHX1B), Ankyrin-B, TGF-beta, CCL13, COL5A1, Necdin, CAP2, MMP-2, MYH14, WNT, FGF13, Collagen III, Collagen IV, HAI-2, IGFBP7/8, S100P, JAM3 organ 3386 1.869E−13 3.750E−11 109 ELF5, Tubby, FGF2, LEKTI, COL5A2, development SPRR1A, p38 MAPK, Syk, IRF6, CARP, Cx30, Keratin 19, BDNF, Plastin, HAS, HEG1, PLA2, Collagen V, G-protein beta, Basonuclin-2, FAM101B, Sciellin, DAB2, CDH1, MAP7(EMAP115), FOG2, Galectin-1, Keratin 16, Pitx3, CCL2, FHL1 (SLIM1), CD24, Galpha(q)-specific peptide GPCRs, Zac1, TAJ(TNFRSF19), Caspr2, Prostasin, PDE, LAMA3 (Epiligrin), E-cadherin, DKK3, TGF-beta 2, Fibrillin 1, COL1A2, SPRR1B, GCNT3, SPRR3, LAMC2, SPTBN(spectrin1-4), DSPP, Maspin, PPL(periplakin), Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, Laminin 5, G-protein beta/gamma, Myoglobin, Nidogen, RHG7, FGFR1, Neuregulin 1, Osteonectin, Vimentin, BVES, ELF3, CTGF, ADAM-TS1, Serglycin, UFO, EPB41L5, MyHC, Lysyl oxidase, PKC, Aquaporin 3, Tcf(Lef), BLNK, SIP1 (ZFHX1B), IGFBP7, G-protein gamma, Ankyrin-B, TCF8, TUB, TGF-beta, HAS2, FA2H, Kallikrein 5, Fibrillin, CCL13, DFNA5, COL5A1, MMP-2, LAMB3, WNT5B, Galpha(t)-specific GPCRs, Keratin 5, Kallikrein 3 (PSA), POU2F3, WNT, FGF13, Collagen III, Collagen IV, HAI-1, ErbB3, IGFBP7/8, M-Ras, Desmoplakin, JAM3 circulatory system 1023 2.201E−13 4.077E−11 52 RECK, FGF2, LAMA4, p38 MAPK, Syk, development CARP, HAS, HEG1, PAI1, Collagen V, COL4A1, FOG2, CCL2, PGAR, Galpha(q)- specific peptide GPCRs, N-cadherin, PDE, TGF-beta 2, Fibrillin 1, COL1A2, Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, Myoglobin, RHG7, FGFR1, Neuregulin 1, Osteonectin, CTGF, ADAM- T51, MyHC, Lysyl oxidase, PKC, Tcf(Lef), Ankyrin-B, TGF-beta, HAS2, Fibrillin, CCL13, PKC-mu, COL5A1, MMP-2, Kallikrein 3 (PSA), WNT, Collagen III, Collagen IV, HAI-1, ErbB3, IGFBP7/8, Desmoplakin, JAM3 cardiovascular 1023 2.201E−13 4.077E−11 52 RECK, FGF2, LAMA4, p38 MAPK, Syk, system CARP, HAS, HEG1, PAI1, Collagen V, development COL4A1, FOG2, CCL2, PGAR, Galpha(q)- specific peptide GPCRs, N-cadherin, PDE, TGF-beta 2, Fibrillin 1, COL1A2, Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, Myoglobin, RHG7, FGFR1, Neuregulin 1, Osteonectin, CTGF, ADAM- TS1, MyHC, Lysyl oxidase, PKC, Tcf(Lef), Ankyrin-B, TGF-beta, HAS2, Fibrillin, CCL13, PKC-mu, COL5A1, MMP-2, Kallikrein 3 (PSA), WNT, Collagen III, Collagen IV, HAI-1, ErbB3, IGFBP7/8, Desmoplakin, JAM3 cellular 1137 3.021E−13 5.388E−11 55 COL5A2, ATR/TEM8, FLRT2, p38 MAPK, component N-chimaerin, CARP, Keratin 19, BDNF, HAS, morphogenesis Ankyrin-G, HEG1, Beta-fodrin, COL6A1, PLA2, Collagen V, FAM101B, COL4A1, DAB2, MAP7(EMAP115), Olfactory receptor, CD24, Galpha(q)-specific peptide GPCRs, UCHL1, R-cadherin, E-cadherin, TGF-beta 2, SPTBN(spectrin1-4), PLA2G10, MAP-1B, Tissue kallikreins, Gremlin, PMP22, FGFR1, Neuregulin 1, BVES, UFO, EPB41L5, MyHC, PKC, Tcf(Lef), CRMP4, Ankyrin-B, TGF- beta, HAS2, Cadherin 11, COL5A1, Kallikrein 8, Necdin, CAP2, MYH14, WNT, Collagen III, Collagen IV, ErbB3, S100B blood vessel 614 3.601E−13 6.193E−11 39 RECK, FGF2, LAMA4, p38 MAPK, Syk, development HAS, HEG1, PAI1, Collagen V, COL4A1, FOG2, CCL2, PGAR, Galpha(q)-specific peptide GPCRs, N-cadherin, PDE, TGF-beta 2, COL1A2, TCF7L2 (TCF4), OVOL2, Gremlin, FGFR1, CTGF, MyHC, Lysyl oxidase, PKC, Tcf(Lef), TGF-beta, HAS2, CCL13, PKC-mu, COL5A1, MMP-2, WNT, Collagen III, Collagen IV, HAI-1, IGFBP7/8, JAM3 neurogenesis 1796 4.204E−13 6.979E−11 72 Tubby, FGF2, LEKTI, COL5A2, FLRT2, N- chimaerin, CARP, BDNF, Ankyrin-G, Beta- fodrin, COL6A1, PLA2, Collagen V, COL4A1, DAB2, Galectin-1, Pitx3, CCL2, Olfactory receptor, CD24, Galpha(q)-specific peptide GPCRs, C1s, UCHL1, FGF5, N- cadherin, Caspr2, DYNLL, PDE, R-cadherin, E-cadherin, TGF-beta 2, SPTBN(spectrin1-4), PLA2G10, MAP-1B, Tissue kallikreins, TCF7L2 (TCF4), ZCCHC24, G-protein beta/gamma, PMP22, FGFR1, Neuregulin 1, Vimentin, UFO, MyHC, PKC, Tcf(Lef), CRMP4, G-protein gamma, Ankyrin-B, TCF8, TGF-beta, FA2H, Neurotractin, Cadherin 11, CCL13, DFNA5, PKC-mu, COL5A1, Kallikrein 8, Necdin, CAP2, MMP-2, MYH14, WNT5B, ITF2, WNT, FGF13, Kallikrein 6 (Neurosin), Collagen III, Collagen IV, ErbB3, S100B cell 1047 5.400E−13 8.667E−11 52 COL5A2, ATR/TEM8, FLRT2, p38 MAPK, morphogenesis N-chimaerin, BDNF, HAS, Ankyrin-G, HEG1, Beta-fodrin, COL6A1, PLA2, Collagen V, FAM101B, COL4A1, DAB2, MAP7(EMAP115), Olfactory receptor, CD24, Galpha(q)-specific peptide GPCRs, UCHL1, R-cadherin, E-cadherin, TGF-beta 2, SPTBN(spectrin1-4), PLA2G10, MAP-1B, Tissue kallikreins, Gremlin, FGFR1, Neuregulin 1, BVES, UFO, EPB41L5, MyHC, PKC, Tcf(Lef), CRMP4, Ankyrin-B, TGF- beta, HAS2, Cadherin 11, COL5A1, Kallikrein 8, Necdin, CAP2, MYH14, WNT, Collagen III, Collagen IV, ErbB3, S100B response to 1775 7.108E−13 1.104E−10 71 Tubby, FGF2, IL13RA2, COL5A2, CDS1, external stimulus FLRT2, p38 MAPK, Syk, Galpha(i)-specific prostanoid GPCRs, N-chimaerin, TBXA2R, CARP, BDNF, Ankyrin-G, Beta-fodrin, COL6A1, PLA2, Collagen V, Calgranulin A, G-protein beta, COL4A1, Calprotectin (S100A8/A9) complex, CCL2, Olfactory receptor, PGAR, Galpha(q)-specific prostanoid GPCRs, LIPE, CD24, Galpha(q)-specific peptide GPCRs, CDP-diacylglycerol synthase, PDE, R-cadherin, TGF-beta 2, Cmtm3, SPTBN(spectrin1-4), PLA2G10, DSPP, MAP- 1B, Tissue kallikreins, Kallikrein 1, G-protein beta/gamma, Gpc6, FGFR1, TRPA1, AP-1 mu subunits, Osteonectin, Vimentin, UFO, MyHC, Calgranulin B, PKC, Aquaporin 3, Tcf(Lef), CRMP4, G-protein gamma, Ankyrin-B, TCF8, TUB, TGF-beta, CCL13, NGAL, COL5A1, CAP2, MMP-2, PTAFR, MYH14, Galpha(t)- specific GPCRs, WNT, NPNT, Collagen III, Collagen IV cellular 5165 7.831E−13 1.178E−10 143 RECK, Tubby, FGF2, LEKTI, COL5A2, component ATR/TEM8, P-cadherin, LAMA4, FLRT2, organization or p38 MAPK, N-chimaerin, Tubulin alpha 1A, biogenesis ITGB6, CARP, Keratin 19, BDNF, Plastin, HAS, Ankyrin-G, NEPH1, P3H2, HEG1, Beta-fodrin, PAI1, COL6A1, Nectin-4, PLA2, Collagen V, OSF-2, Synaptotagmin VII, FAM101B, TMSL8, CPA4, FGD2, COL4A1, MARVELD3, Calprotectin (S100A8/A9) complex, DAB2, CDH1, MAP7(EMAP115), Keratin 16, GLT25D2, CCL2, SRPX, Olfactory receptor, PGAR, KIAA1043, NAP1L3, LIPE, PAR1, CD24, Galpha(q)- specific peptide GPCRs, UCHL1, Tubulin alpha, Synaptotagmin, N-cadherin, Caspr2, PRR5, DYNLL, PDE, R-cadherin, LAMA3 (Epiligrin), E-cadherin, FAT4, TGF-beta 2, HOOK1, Fibrillin 1, Collagen XII, COL1A2, LAMC2, Tricellulin, SPTBN(spectrin1-4), PLA2G10, MAP-1B, Chk2, Maspin, Tissue kallikreins, TCF7L2 (TCF4), Gremlin, DLC1 (Dynein LC8a), Laminin 5, SHPS-1, PMP22, Nidogen, RHG7, FGFR1, Rab-25, LyGDI, Neuregulin 1, AP-1 mu subunits, Osteonectin, Vimentin, BVES, ELF3, CTGF, Serglycin, ATP8B3, UFO, EPB41L5, MyHC, Lysyl oxidase, Calgranulin B, PKC, FR-alpha, Tcf(Lef), WaspIP, MPP7, CRMP4, Ankyrin- B, PAK6, TGF-beta, HAS2, FA2H, Fibrillin, Cadherin 11, CCL13, NGAL, PKC-mu, M- cadherin, CLIP3, COL5A1, Kallikrein 8, Necdin, CAP2, MMP-2, LAMB3, MYH14, Keratin 5, ITF2, WNT, F16P, FGF13, NPNT, Collagen III, Collagen IV, ChAF1 subunit B, HAI-1, ErbB3, IGFBP7/8, M-Ras, Mucin 20, S100B, Desmoplakin locomotion 1363 1.218E−12 1.777E−10 60 FGF2, COL5A2, FLRT2, p38 MAPK, Syk, N- chimaerin, BDNF, Ankyrin-G, Beta-fodrin, COL6A1, PLA2, Collagen V, Calgranulin A, COL4A1, Matriptase, Calprotectin (S100A8/A9) complex, CCL2, Olfactory receptor, CD24, Galpha(q)-specific peptide GPCRs, N-cadherin, PDE, R-cadherin, TGF- beta 2, Cmtm3, COL1A2, SPTBN(spectrin1-4), PLA2G10, OVOL2, Gremlin, SHPS-1, Gpc6, FGFR1, Neuregulin 1, CTGF, UFO, EPB41L5, MyHC, Calgranulin B, PKC, Tcf(Lef), CRMP4, SIP1 (ZFHX1B), Ankyrin- B, TGF-beta, CCL13, COL5A1, Necdin, CAP2, MMP-2, PTAFR, MYH14, Galpha(t)- specific GPCRs, WNT, FGF13, Collagen III, Collagen IV, IGFBP7/8, S100P, JAM3 vasculature 652 2.331E−12 3.106E−10 39 RECK, FGF2, LAMA4, p38 MAPK, Syk, development HAS, HEG1, PAI1, Collagen V, COL4A1, FOG2, CCL2, PGAR, Galpha(q)-specific peptide GPCRs, N-cadherin, PDE, TGF-beta 2, COL1A2, TCF7L2 (TCF4), OVOL2, Gremlin, FGFR1, CTGF, MyHC, Lysyl oxidase, PKC, Tcf(Lef), TGF-beta, HAS2, CCL13, PKC-mu, COL5A1, MMP-2, WNT, Collagen III, Collagen IV, HAI-1, IGFBP7/8, JAM3 regulation of 2241 2.383E−12 3.106E−10 81 Tubby, FGF2, LEKTI, LAMA4, FLRT2, developmental INPP4B, p38 MAPK, Syk, Galpha(i)-specific process prostanoid GPCRs, N-chimaerin, TBXA2R, CARP, BDNF, PTGIS, MBNL3, PAI1, PLA2, FGD2, DAB2, CDH1, FOG2, Galectin-1, CCL2, PGAR, Galpha(q)-specific prostanoid GPCRs, CD24, Galpha(q)-specific peptide GPCRs, IL1RN, N-cadherin, PDE, R-cadherin, LAMA3 (Epiligrin), E-cadherin, TGF-beta 2, SPRR1B, SPRR3, PLA2G10, MAP-1B, Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, ZCCHC24, Laminin 5, PMP22, RHG7, FGFR1, Neuregulin 1, Vimentin, BVES, CTGF, ADAM-TS1, Serglycin, UFO, EPB41L5, MyHC, PKC, Aquaporin 3, Tcf(Lef), CRMP4, TCF8, TGF-beta, Neurotractin, Fibrillin, CCL13, PKC-mu, M- cadherin, Kallikrein 8, MYH14, WNT5B, Kallikrein 3 (PSA), ITF2, WNT, FGF13, NPNT, Kallikrein 6 (Neurosin), Collagen III, RBM24, Collagen IV, IGFBP7/8, S100B wound healing 846 2.394E−12 3.106E−10 45 FGF2, P-cadherin, p38 MAPK, Syk, Galpha(i)-specific prostanoid GPCRs, TBXA2R, PAI1, PLA2, Collagen V, Calgranulin A, Synaptotagmin VII, G-protein beta, Calprotectin (S100A8/A9) complex, FOG2, Galpha(q)-specific prostanoid GPCRs, PAR1, Galpha(q)-specific peptide GPCRs, Tubulin alpha, Synaptotagmin, PDE, TGF-beta 2, COL1A2, SPRR3, Tissue kallikreins, SHPS-1, G-protein beta/gamma, Neuregulin 1, Osteonectin, Vimentin, Serglycin, UFO, MyHC, Lysyl oxidase, PKC, G-protein gamma, TGF-beta, COL5A1, WNT5B, POU2F3, WNT, Kallikrein 6 (Neurosin), Collagen III, ErbB3, Desmoplakin, JAM3 regulation of 1780 2.396E−12 3.106E−10 70 Tubby, FGF2, LEKTI, LAMA4, FLRT2, multicellular INPP4B, p38 MAPK, Syk, Galpha(i)-specific organismal prostanoid GPCRs, N-chimaerin, TBXA2R, development CARP, BDNF, PTGIS, MBNL3, PAI1, PLA2, DAB2, FOG2, Galectin-1, CCL2, PGAR, Galpha(q)-specific prostanoid GPCRs, CD24, Galpha(q)-specific peptide GPCRs, IL1RN, N- cadherin, PDE, R-cadherin, LAMA3 (Epiligrin), E-cadherin, TGF-beta 2, MAP-1B, Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, ZCCHC24, Laminin 5, PMP22, FGFR1, Neuregulin 1, Vimentin, CTGF, ADAM-TS1, Serglycin, UFO, EPB41L5, MyHC, PKC, Aquaporin 3, Tcf(Lef), CRMP4, TCF8, TGF-beta, Neurotractin, Fibrillin, CCL13, PKC-mu, Kallikrein 8, Kallikrein 3 (PSA), ITF2, WNT, FGF13, Kallikrein 6 (Neurosin), Collagen III, RBM24, Collagen IV, IGFBP7/8, S100B chemotaxis 748 2.451E−12 3.106E−10 42 FGF2, COL5A2, FLRT2, p38 MAPK, Syk, N- chimaerin, BDNF, Ankyrin-G, Beta-fodrin, COL6A1, PLA2, Collagen V, Calgranulin A, COL4A1, Calprotectin (S100A8/A9) complex, CCL2, Olfactory receptor, CD24, Galpha(q)- specific peptide GPCRs, PDE, R-cadherin, TGF-beta 2, Cmtm3, SPTBN(spectrin1-4), PLA2G10, FGFR1, MyHC, Calgranulin B, PKC, Tcf(Lef), CRMP4, Ankyrin-B, TGF- beta, CCL13, COL5A1, CAP2, PTAFR, MYH14, Galpha(t)-specific GPCRs, WNT, Collagen III, Collagen IV taxis 750 2.673E−12 3.300E−10 42 FGF2, COL5A2, FLRT2, p38 MAPK, Syk, N- chimaerin, BDNF, Ankyrin-G, Beta-fodrin, COL6A1, PLA2, Collagen V, Calgranulin A, COL4A1, Calprotectin (S100A8/A9) complex, CCL2, Olfactory receptor, CD24, Galpha(q)- specific peptide GPCRs, PDE, R-cadherin, TGF-beta 2, Cmtm3, SPTBN(spectrin1-4), PLA2G10, FGFR1, MyHC, Calgranulin B, PKC, Tcf(Lef), CRMP4, Ankyrin-B, TGF- beta, CCL13, COL5A1, CAP2, PTAFR, MYH14, Galpha(t)-specific GPCRs, WNT, Collagen III, Collagen IV multicellular 116 6.070E−12 7.307E−10 17 Tubby, COL5A2, P3H2, COL6A1, PLA2, organismal Collagen V, COL4A1, Collagen XII, metabolic process COL1A2, Tissue kallikreins, PKC, TUB, COL5A1, MMP-2, Kallikrein 6 (Neurosin), Collagen III, Collagen IV single-organism 4620 6.248E−12 7.338E−10 130 ELF5, RECK, Tubby, FGF2, LEKTI, developmental COL5A2, ATR/TEM8, LAMA4, FLRT2, p38 process MAPK, Syk, N-chimaerin, IRF6, CARP, Cx30, Keratin 19, BDNF, Plastin, HAS, Ankyrin-G, HEG1, Beta-fodrin, PAI1, COL6A1, PLA2, Collagen V, G-protein beta, Basonuclin-2, FAM101B, COL4A1, Sciellin, DAB2, CDH1, MAP7(EMAP115), FOG2, Galectin-1, Pitx3, CCL2, Olfactory receptor, FHL1 (SLIM1), PGAR, CD24, Galpha(q)- specific peptide GPCRs, Zac1, UCHL1, N- cadherin, Desmocollin 3, Caspr2, DYNLL, PDE, R-cadherin, E-cadherin, DKK3, TGF- beta 2, HOOK1, Fibrillin 1, COL1A2, GCNT3, SPTBN(spectrin1-4), PLA2G10, DSPP, MAP-1B, Chk2, Maspin, Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, G-protein beta/gamma, Myoglobin, PMP22, Nidogen, RHG7, FGFR1, Neuregulin 1, AP-1 mu subunits, Osteonectin, Vimentin, BVES, ELF3, CTGF, ADAM-TS1, UFO, EPB41L5, MyHC, Lysyl oxidase, PKC, Aquaporin 3, Tcf(Lef), BLNK, CRMP4, SIP1 (ZFHX1B), IGFBP7, G-protein gamma, Ankyrin-B, LAF4, TCF8, TUB, TGF-beta, HAS2, FA2H, Fibrillin, Cadherin 11, CCL13, DFNA5, PKC-mu, M-cadherin, COL5A1, Kallikrein 8, GRHL2, Necdin, CAP2, MMP-2, MYH14, WNT5B, Galpha(t)-specific GPCRs, Kallikrein 3 (PSA), WNT, FGF13, NPNT, Kallikrein 6 (Neurosin), Collagen III, Collagen IV, HAI-1, ErbB3, IGFBP7/8, M-Ras, S100B, Desmoplakin, JAM3 cell junction 237 7.591E−12 8.702E−10 23 P-cadherin, HEG1, Nectin-4, MARVELD3, N- organization cadherin, R-cadherin, LAMA3 (Epiligrin), E- cadherin, TGF-beta 2, LAMC2, Tricellulin, Laminin 5, RHG7, PKC, MPP7, Ankyrin-B, TGF-beta, Cadherin 11, M-cadherin, LAMB3, Keratin 5, WNT, Desmoplakin cell development 1875 9.467E−12 1.060E−09 71 Tubby, FGF2, COL5A2, ATR/TEM8, FLRT2, N-chimaerin, IRF6, CARP, Keratin 19, BDNF, HAS, Ankyrin-G, HEG1, Beta-fodrin, COL6A1, PLA2, Collagen V, FAM101B, COL4A1, DAB2, MAP7(EMAP115), Pitx3, Olfactory receptor, CD24, Galpha(q)-specific peptide GPCRs, UCHL1, Caspr2, DYNLL, PDE, R-cadherin, E-cadherin, TGF-beta 2, HOOK1, SPTBN(spectrin1-4), PLA2G10, MAP-1B, Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, G-protein beta/gamma, FGFR1, Neuregulin 1, Vimentin, BVES, UFO, EPB41L5, MyHC, PKC, Tcf(Lef), CRMP4, SIP1 (ZFHX1B), G-protein gamma, Ankyrin- B, TGF-beta, HAS2, FA2H, Cadherin 11, COL5A1, Kallikrein 8, Necdin, CAP2, MMP- 2, MYH14, WNT5B, WNT, Collagen III, Collagen IV, ErbB3, S100B, JAM3 regulation of cell 416 9.894E−12 1.083E−09 30 LEKTI, LAMA4, Syk, EDIL3, HAS, Ankyrin- adhesion G, PAI1, DAB2, Galectin-1, CD24, Galpha(q)- specific peptide GPCRs, IL1RN, PDE, LAMA3 (Epiligrin), E-cadherin, TGF-beta 2, Gremlin, Laminin 5, Nidogen, LyGDI, Neuregulin 1, EPB41L5, PKC, Tcf(Lef), TGF- beta, HAS2, MMP-2, WNT, NPNT, ErbB3 multicellular 105 1.393E−11 1.490E−09 16 Tubby, COL5A2, P3H2, COL6A1, Collagen organismal V, COL4A1, Collagen XII, COL1A2, Tissue macromolecule kallikreins, PKC, TUB, COL5A1, MMP-2, metabolic process Kallikrein 6 (Neurosin), Collagen III, Collagen IV generation of 1700 2.075E−11 2.172E−09 66 Tubby, LEKTI, COL5A2, FLRT2, N- neurons chimaerin, CARP, BDNF, Ankyrin-G, Beta- fodrin, COL6A1, PLA2, Collagen V, COL4A1, DAB2, Galectin-1, Pitx3, Olfactory receptor, CD24, Galpha(q)-specific peptide GPCRs, UCHL1, N-cadherin, Caspr2, DYNLL, PDE, R-cadherin, E-cadherin, TGF- beta 2, SPTBN(spectrin1-4), PLA2G10, MAP- 1B, Tissue kallikreins, TCF7L2 (TCF4), ZCCHC24, G-protein beta/gamma, PMP22, FGFR1, Neuregulin 1, Vimentin, UFO, MyHC, PKC, Tcf(Lef), CRMP4, G-protein gamma, Ankyrin-B, TCF8, TGF-beta, Neurotractin, Cadherin 11, DFNA5, PKC-mu, COL5A1, Kallikrein 8, Necdin, CAP2, MMP- 2, MYH14, WNT5B, ITF2, WNT, FGF13, Kallikrein 6 (Neurosin), Collagen III, Collagen IV, ErbB3, S100B regulation of 903 2.148E−11 2.201E−09 45 FGF2, LEKTI, p38 MAPK, Galpha(i)-specific anatomical prostanoid GPCRs, N-chimaerin, TBXA2R, structure BDNF, PTGIS, PAI1, FGD2, DAB2, CCL2, morphogenesis PGAR, Galpha(q)-specific prostanoid GPCRs, Galpha(q)-specific peptide GPCRs, IL1RN, N- cadherin, PDE, R-cadherin, E-cadherin, TGF- beta 2, SPRR1B, SPRR3, MAP-1B, Tissue kallikreins, TCF7L2 (TCF4), Gremlin, RHG7, FGFR1, Vimentin, BVES, ADAM-TS1, EPB41L5, MyHC, PKC, Tcf(Lef), TGF-beta, CCL13, PKC-mu, MYH14, Kallikrein 3 (PSA), WNT, FGF13, Collagen IV, S100B organ 1057 3.234E−11 3.244E−09 49 Tubby, FGF2, COL5A2, p38 MAPK, Syk, morphogenesis CARP, Cx30, HAS, HEG1, Collagen V, Basonuclin-2, FOG2, Pitx3, CCL2, FHL1 (SLIM1), Galpha(q)-specific peptide GPCRs, PDE, TGF-beta 2, COL1A2, GCNT3, DSPP, Maspin, Tissue kallikreins, TCF7L2 (TCF4), OVOL2, Gremlin, G-protein beta/gamma, RHG7, FGFR1, Neuregulin 1, CTGF, ADAM- TS1, MyHC, PKC, Aquaporin 3, Tcf(Lef), G- protein gamma, TCF8, TGF-beta, HAS2, Fibrillin, COL5A1, MMP-2, WNT5B, Kallikrein 3 (PSA), WNT, ErbB3, IGFBP7/8, Desmoplakin epidermis 358 4.079E−11 4.008E−09 27 LEKTI, SPRR1A, IRF6, Sciellin, Keratin 16, development TAJ(TNFRSF19), Prostasin, LAMA3 (Epiligrin), TGF-beta 2, SPRR1B, SPRR3, LAMC2, PPL(periplakin), Tissue kallikreins, Laminin 5, FGFR1, ELF3, CTGF, TGF-beta, FA2H, Kallikrein 5, LAMB3, Keratin 5, POU2F3, WNT, IGFBP7/8, Desmoplakin positive regulation 1293 4.627E−11 4.456E−09 55 AKAP12, RECK, FGF2, p38 MAPK, Syk, of signaling CARP, PTGIS, PLA2, Calgranulin A, Calprotectin (S100A8/A9) complex, DAB2, Galectin-1, CCL2, SRPX, PAR1, MAPBPIP, CD24, Galpha(q)-specific peptide GPCRs, IL1RN, BPGAP1, N-cadherin, TAJ(TNFRSF19), PRR5, PDE, PRR5- ARHGAP8, TGF-beta 2, FAM110C, Tissue kallikreins, TCF7L2 (TCF4), G-protein beta/gamma, FGFR1, HBP17, Neuregulin 1, BVES, CTGF, UFO, Calgranulin B, PKC, Tcf(Lef), MPP7, SIP1 (ZFHX1B), G-protein gamma, TGF-beta, Kallikrein 5, CCL13, DFNA5, PKC-mu, WNT, NPNT, Kallikrein 6 (Neurosin), Collagen III, ErbB3, IGFBP7/8, Mucin 20, S100B

TABLE 12B PC1 GO Processes In Network Objects from Processes Total p-value FDR Data Active Data axonogenesis 616 2.613E−09 4.905E−06 29 Ephrin-B receptors, FLRT2, FEZ1, Tubulin beta, Neuropilin-2, PLA2, SLIT3, Galpha(q)-specific peptide GPCRs, UCHL1, Kalirin, SHH, Ephrin-B receptor 2, MAP-1B, SLIT2, ERM proteins, Neuropilin-1, Ephrin-B receptor 1, Cofilin, SRGAP2, MyHC, PKC, CRMP4, PTPR-mu, Hedgehog, CAP2, Necdin, MYH10, TRPC1, MYH14 cell 656 2.671E−09 4.905E−06 30 Ephrin-B receptors, FLRT2, FEZ1, Tubulin beta, morphogenesis Neuropilin-2, PLA2, SLIT3, Galpha(q)-specific involved in peptide GPCRs, UCHL1, Kalirin, SHH, Ephrin-B neuron receptor 2, MAP-1B, Syndecan-2, SLIT2, ERM differentiation proteins, Neuropilin-1, Ephrin-B receptor 1, Cofilin, SRGAP2, MyHC, PKC, CRMP4, PTPR-mu, Hedgehog, CAP2, Necdin, MYH10, TRPC1, MYH14 cell 792 4.113E−09 4.905E−06 33 SLUG, Ephrin-B receptors, FLRT2, FEZ1, Tubulin morphogenesis beta, Neuropilin-2, PLA2, SLIT3, Galpha(q)- involved in specific peptide GPCRs, Willin, UCHL1, Kalirin, differentiation SHH, Ephrin-B receptor 2, MAP-1B, Syndecan-2, SLIT2, ERM proteins, Neuropilin-1, Ephrin-B receptor 1, Cofilin, SRGAP2, MyHC, PKC, CRMP4, PTPR-mu, Hedgehog, CAP2, Necdin, MYH10, TRPC1, MYH14, BASP1 neuron 679 5.898E−09 4.905E−06 30 Ephrin-B receptors, FLRT2, FEZ1, Tubulin beta, projection Neuropilin-2, PLA2, SLIT3, Galpha(q)-specific morphogenesis peptide GPCRs, UCHL1, Kalirin, SHH, Ephrin-B receptor 2, MAP-1B, Syndecan-2, SLIT2, ERM proteins, Neuropilin-1, Ephrin-B receptor 1, Cofilin, SRGAP2, MyHC, PKC, CRMP4, PTPR-mu, Hedgehog, CAP2, Necdin, MYH10, TRPC1, MYH14 neuron 489 6.883E−09 4.905E−06 25 Ephrin-B receptors, FLRT2, FEZ1, Tubulin beta, projection Neuropilin-2, PLA2, SLIT3, Galpha(q)-specific guidance peptide GPCRs, SHH, Ephrin-B receptor 2, SLIT2, ERM proteins, Neuropilin-1, Ephrin-B receptor 1, Cofilin, SRGAP2, MyHC, PKC, CRMP4, PTPR- mu, Hedgehog, CAP2, MYH10, TRPC1, MYH14 axon guidance 489 6.883E−09 4.905E−06 25 Ephrin-B receptors, FLRT2, FEZ1, Tubulin beta, Neuropilin-2, PLA2, SLIT3, Galpha(q)-specific peptide GPCRs, SHH, Ephrin-B receptor 2, SLIT2, ERM proteins, Neuropilin-1, Ephrin-B receptor 1, Cofilin, SRGAP2, MyHC, PKC, CRMP4, PTPR- mu, Hedgehog, CAP2, MYH10, TRPC1, MYH14 single- 4620 1.073E−08 6.553E−06 101 SNF2L1, PAR2, c-Maf, QKI, RECK, SLUG, Alpha organism crystallin B, IQCJ-SCHIP1, AEBP1, Ephrin-B developmental receptors, FLRT2, Caspase-6, SLC29A2, PEDF process (serpinF1), BDH, Axin, FEZ1, Tubulin beta, Neuropilin-2, DUOX1, PLA2, LEC1, GCR-alpha, SLAC2-B, Axin2, SLIT3, TIMP2, Plakophilin 2, MAP7(EMAP115), FOG2, Calpain 5, Galectin-1, Myosin Va, SCHIP1, Galpha(q)-specific peptide GPCRs, Epsilon-sarcoglycan, Willin, UCHL1, Kalirin, SHH, DAZAP, Ephrin-B receptor 2, Decorin proteoglycan, CDX1, HMGI/Y, DUOX2, NAT-1, LDB2, MAP-1B, Chk2, HOXB9, Syndecan-2, GCR-beta, SLIT2, Polycystin 2, NNMT, ERM proteins, DZIP, Neuropilin-1, NKCC1, Ephrin-B receptor 1, AP-1 mu subunits, HNF4-alpha, Cofilin, c-Myb, DMP4, Alpha/epsilon-sarcoglycan, AML1/TRPS1 fusion protein, NOX1, Vimentin, Osteonectin, SRGAP2, MyHC, PKC, CRMP4, MGP, PTPR-mu, G-protein gamma, Hedgehog, FA2H, SLC5A1, DFNA5, AKT(PKB), PKC-mu, RILP (REST-interacting LIM domain protein), CAP2, Necdin, ECM2/SC1, MYH10, TRPC1, MYH14, TAZ, NAT-2, KCNQ1, BASP1, HNF4, STRBP, PRKD2, M-Ras, Decorin, JAM3 system 4665 1.817E−08 9.713E−06 101 SNF2L1, PAR2, c-Maf, QKI, RECK, SLUG, Alpha development crystallin B, IQCJ-SCHIP1, AEBP1, Ephrin-B receptors, FLRT2, Caspase-6, SLC29A2, LZTS1, PEDF (serpinF1), BDH, Axin, FEZ1, Tubulin beta, Neuropilin-2, PLA2, LEC1, GCR-alpha, MUPP1, SLAC2-B, ERR1, SPG20, Axin2, SLIT3, TIMP2, Plakophilin 2, MAP7(EMAP115), FOG2, Calpain 5, Galectin-1, Myosin Va, SCHIP1, Galpha(q)-specific peptide GPCRs, Epsilon-sarcoglycan, UCHL1, Kalirin, SHH, DAZAP, Ephrin-B receptor 2, Decorin proteoglycan, CDX1, DUOX2, NAT-1, LDB2, MAP-1B, HOXB9, Syndecan-2, GCR-beta, SLIT2, Glis2, Polycystin 2, NNMT, ERM proteins, Neuropilin-1, ENC1, NKCC1, Ephrin-B receptor 1, HNF4-alpha, Cofilin, c-Myb, DMP4, Alpha/epsilon-sarcoglycan, AML1/TRPS1 fusion protein, NOX1, Vimentin, Osteonectin, SRGAP2, MyHC, PKC, CRMP4, MGP, PTPR-mu, G-protein gamma, Hedgehog, FA2H, SLC5A1, DFNA5, AKT(PKB), PKC-mu, NELL1, RILP (REST- interacting LIM domain protein), CAP2, Necdin, MYH10, TRPC1, MYH14, TAZ, NAT-2, KCNQ1, TRPS1, BASP1, HNF4, PRKD2, M-Ras, Decorin, JAM3 axon 680 2.340E−08 1.112E−05 29 Ephrin-B receptors, FLRT2, FEZ1, Tubulin beta, development Neuropilin-2, PLA2, SLIT3, Galpha(q)-specific peptide GPCRs, UCHL1, Kalirin, SHH, Ephrin-B receptor 2, MAP-1B, SLIT2, ERM proteins, Neuropilin-1, Ephrin-B receptor 1, Cofilin, SRGAP2, MyHC, PKC, CRMP4, PTPR-mu, Hedgehog, CAP2, Necdin, MYH10, TRPC1, MYH14 establishment 139 3.577E−08 1.473E−05 13 Ephrin-B receptors, MAP7(EMAP115), SHH, or MAP-1B, ERM proteins, Ephrin-B receptor 1, maintenance Cofilin, MyHC, PKC, Hedgehog, AMOTL1 (Jeap), of cell polarity CAP2, JAM3 regulation of 457 3.789E−08 1.473E−05 23 PAR2, Ephrin-B receptors, LZTS1, FEZ1, GCR- cell projection alpha, SPG20, Galectin-1, CDC42EP5, Galpha(q)- organization specific peptide GPCRs, Kalirin, Ephrin-B receptor 2, MAP-1B, Syndecan-2, GCR-beta, SLIT2, Neuropilin-1, HNF4-alpha, Vimentin, PKC, CRMP4, AKT(PKB), PKC-mu, HNF4 cell projection 831 4.588E−08 1.635E−05 32 Ephrin-B receptors, FLRT2, FEZ1, Tubulin beta, morphogenesis Neuropilin-2, PLA2, SLIT3, Galpha(q)-specific peptide GPCRs, UCHL1, Kalirin, SHH, Ephrin-B receptor 2, MAP-1B, Syndecan-2, SLIT2, ERM proteins, DZIP, Neuropilin-1, Ephrin-B receptor 1, Cofilin, SRGAP2, MyHC, PKC, CRMP4, PTPR- mu, Hedgehog, CAP2, Necdin, MYH10, TRPC1, MYH14, TAZ anatomical 2492 6.023E−08 1.981E−05 64 QKI, SLUG, Alpha crystallin B, IQCJ-SCHIP1, structure Ephrin-B receptors, FLRT2, Axin, FEZ1, Tubulin morphogenesis beta, Neuropilin-2, PLA2, GCR-alpha, Axin2, SLIT3, Plakophilin 2, MAP7(EMAP115), FOG2, Myosin Va, SCHIP1, Galpha(q)-specific peptide GPCRs, Willin, UCHL1, Kalirin, SHH, Ephrin-B receptor 2, Decorin proteoglycan, CDX1, DUOX2, MAP-1B, Syndecan-2, GCR-beta, SLIT2, Polycystin 2, ERM proteins, DZIP, Neuropilin-1, NKCC1, Ephrin-B receptor 1, HNF4-alpha, Cofilin, DMP4, AML1/TRPS1 fusion protein, NOX1, SRGAP2, MyHC, PKC, CRMP4, MGP, PTPR-mu, G-protein gamma, Hedgehog, PKC-mu, RILP (REST-interacting LIM domain protein), CAP2, Necdin, MYH10, TRPC1, MYH14, TAZ, BASP1, HNF4, PRKD2, Decorin, JAM3 cell part 855 8.804E−08 2.689E−05 32 Ephrin-B receptors, FLRT2, FEZ1, Tubulin beta, morphogenesis Neuropilin-2, PLA2, SLIT3, Galpha(q)-specific peptide GPCRs, UCHL1, Kalirin, SHH, Ephrin-B receptor 2, MAP-1B, Syndecan-2, SLIT2, ERM proteins, DZIP, Neuropilin-1, Ephrin-B receptor 1, Cofilin, SRGAP2, MyHC, PKC, CRMP4, PTPR- mu, Hedgehog, CAP2, Necdin, MYH10, TRPC1, MYH14, TAZ regulation of 903 9.475E−08 2.701E−05 33 SLUG, Ephrin-B receptors, Caspase-6, LZTS1, anatomical PEDF (serpinF1), Axin, SPG20, Axin2, structure CDC42EP5, Galpha(q)-specific peptide GPCRs, morphogenesis SHH, Ephrin-B receptor 2, DDAH1, MAP-1B, Syndecan-2, SLIT2, ERM proteins, Neuropilin-1, HNF4-alpha, Cofilin, AML1/TRPS1 fusion protein, Vimentin, MyHC, PKC, PTPR-mu, Hedgehog, PKC-mu, MYH10, MYH14, TAZ, BASP1, HNF4, PRKD2 cell 1047 1.065E−07 2.845E−05 36 SLUG, Ephrin-B receptors, FLRT2, FEZ1, Tubulin morphogenesis beta, Neuropilin-2, PLA2, SLIT3, MAP7(EMAP115), Galpha(q)-specific peptide GPCRs, Willin, UCHL1, Kalirin, SHH, Ephrin-B receptor 2, MAP-1B, Syndecan-2, SLIT2, ERM proteins, DZIP, Neuropilin-1, Ephrin-B receptor 1, Cofilin, SRGAP2, MyHC, PKC, CRMP4, PTPR- mu, Hedgehog, CAP2, Necdin, MYH10, TRPC1, MYH14, TAZ, BASP1 chemotaxis 748 1.789E−07 4.354E−05 29 CCL28, Ephrin-B receptors, FLRT2, FEZ1, Tubulin beta, Neuropilin-2, PLA2, SLIT3, Galpha(q)- specific peptide GPCRs, SHH, Ephrin-B receptor 2, Cmtm3, HOXB9, SLIT2, ERM proteins, Neuropilin-1, Ephrin-B receptor 1, Cofilin, SRGAP2, MyHC, PKC, CMTM4, CRMP4, PTPR- mu, Hedgehog, CAP2, MYH10, TRPC1, MYH14 taxis 750 1.892E−07 4.354E−05 29 CCL28, Ephrin-B receptors, FLRT2, FEZ1, Tubulin beta, Neuropilin-2, PLA2, SLIT3, Galpha(q)- specific peptide GPCRs, SHH, Ephrin-B receptor 2, Cmtm3, HOXB9, SLIT2, ERM proteins, Neuropilin-1, Ephrin-B receptor 1, Cofilin, SRGAP2, MyHC, PKC, CMTM4, CRMP4, PTPR- mu, Hedgehog, CAP2, MYH10, TRPC1, MYH14 establishment 87 1.998E−07 4.354E−05 10 Ephrin-B receptors, SHH, MAP-1B, Ephrin-B of cell polarity receptor 1, Cofilin, MyHC, PKC, Hedgehog, AMOTL1 (Jeap), JAM3 cellular 1526 2.037E−07 4.354E−05 45 PAR2, SLUG, IQCJ-SCHIP1, Ephrin-B receptors, component FLRT2, FEZ1, Tubulin beta, Tensin, Neuropilin-2, movement PLA2, Matriptase, SLIT3, Myosin Va, SCHIP1, Galpha(q)-specific peptide GPCRs, Willin, UCHL1, SHH, Ephrin-B receptor 2, Tubulin beta 6, MAP- 1B, MSN (moesin), HOXB9, Caldesmon, SLIT2, ERM proteins, Neuropilin-1, Ephrin-B receptor 1, Cofilin, NOX1, Vimentin, SRGAP2, MyHC, PKC, CRMP4, PTPR-mu, Hedgehog, AMOTL1 (Jeap), CAP2, Necdin, MYH10, TRPC1, MYH14, STRBP, JAM3 multicellular 5406 3.490E−07 7.105E−05 108 SNF2L1, PAR2, c-Maf, QKI, RECK, SLUG, Alpha organismal crystallin B, IQCJ-SCHIP1, AEBP1, Ephrin-B development receptors, FLRT2, Caspase-6, SLC29A2, LZTS1, PEDF (serpinF1), BDH, Axin, FEZ1, Tubulin beta, Neuropilin-2, DUOX1, PLA2, LEC1, GCR-alpha, MUPP1, SLAC2-B, ERR1, SPG20, Axin2, SLIT3, TIMP2, Plakophilin 2, MAP7(EMAP115), FOG2, Calpain 5, Galectin-1, Myosin Va, SCHIP1, Galpha(q)-specific peptide GPCRs, Epsilon- sarcoglycan, UCHL1, PAQR8, Kalirin, SHH, DAZAP, Ephrin-B receptor 2, Decorin proteoglycan, CDX1, FRAT2, DUOX2, TMPRSS2/ERG fusion protein, NAT-1, LDB2, MAP-1B, HOXB9, Syndecan-2, GCR-beta, SLIT2, Glis2, Polycystin 2, NNMT, ERM proteins, DZIP, Neuropilin-1, ENC1, NKCC1, Ephrin-B receptor 1, HNF4-alpha, Cofilin, c-Myb, DMP4, Alpha/epsilon-sarcoglycan, AML1/TRPS1 fusion protein, NOX1, Vimentin, Osteonectin, SRGAP2, MyHC, PKC, CRMP4, MGP, PTPR-mu, G-protein gamma, Hedgehog, FA2H, SLC5A1, DFNA5, AKT(PKB), PKC-mu, NELL1, RILP (REST- interacting LIM domain protein), CAP2, Necdin, ECM2/SC1, MYH10, TRPC1, MYH14, TAZ, NAT-2, KCNQ1, TRPS1, BASP1, HNF4, STRBP, PRKD2, M-Ras, Decorin, JAM3 retinal 23 3.907E−07 7.594E−05 6 Ephrin-B receptors, Ephrin-B receptor 2, SLIT2, ganglion cell Neuropilin-1, Ephrin-B receptor 1, PTPR-mu axon guidance regulation of 1780 4.217E−07 7.841E−05 49 c-Maf, SLUG, Ephrin-B receptors, FLRT2, multicellular Caspase-6, LZTS1, PEDF (serpinF1), Axin, FEZ1, organismal PLA2, GCR-alpha, ERR1, SPG20, Axin2, TIMP2, development FOG2, Galectin-1, Galpha(q)-specific peptide GPCRs, Kalirin, SHH, Ephrin-B receptor 2, DDAH1, MAP-1B, Syndecan-2, GCR-beta, SLIT2, Neuropilin-1, Ephrin-B receptor 1, HNF4-alpha, c- Myb, DMP4, AML1/TRPS1 fusion protein, Vimentin, SRGAP2, MyHC, PKC, CRMP4, MGP, PTPR-mu, Hedgehog, AKT(PKB), PKC-mu, NELL1, RILP (REST-interacting LIM domain protein), TAZ, TRPS1, BASP1, HNF4, PRKD2 locomotion 1363 4.618E−07 8.209E−05 41 PAR2, SLUG, CCL28, IQCJ-SCHIP1, Ephrin-B receptors, FLRT2, FEZ1, Tubulin beta, Tensin, Neuropilin-2, PLA2, Matriptase, SLIT3, Myosin Va, SCHIP1, Galpha(q)-specific peptide GPCRs, SHH, Ephrin-B receptor 2, Cmtm3, MSN (moesin), HOXB9, SLIT2, ERM proteins, Neuropilin-1, Ephrin-B receptor 1, Cofilin, NOX1, SRGAP2, MyHC, PKC, CMTM4, CRMP4, PTPR-mu, Hedgehog, AMOTL1 (Jeap), CAP2, Necdin, MYH10, TRPC1, MYH14, JAM3 ameboidal cell 203 4.799E−07 8.209E−05 14 IQCJ-SCHIP1, Ephrin-B receptors, Tensin, migration Neuropilin-2, SCHIP1, Galpha(q)-specific peptide GPCRs, SHH, SLIT2, Neuropilin-1, Cofilin, SRGAP2, MyHC, Hedgehog, AMOTL1 (Jeap) anatomical 5314 5.334E−07 8.554E−05 106 SNF2L1, PAR2, c-Maf, QKI, RECK, SLUG, Alpha structure crystallin B, IQCJ-SCHIP1, AEBP1, Ephrin-B development receptors, FLRT2, Caspase-6, SLC29A2, LZTS1, PEDF (serpinF1), BDH, Axin, FEZ1, Tubulin beta, Neuropilin-2, DUOX1, PLA2, LEC1, GCR-alpha, MUPP1, SLAC2-B, ERR1, SPG20, Axin2, SLIT3, TIMP2, Plakophilin 2, MAP7(EMAP115), FOG2, Calpain 5, Galectin-1, Myosin Va, SCHIP1, Galpha(q)-specific peptide GPCRs, Epsilon- sarcoglycan, Willin, UCHL1, Kalirin, SHH, DAZAP, Ephrin-B receptor 2, Decorin proteoglycan, CDX1, DUOX2, NAT-1, LDB2, MAP-1B, HOXB9, Syndecan-2, GCR-beta, SLIT2, Glis2, Polycystin 2, NNMT, ERM proteins, DZIP, Neuropilin-1, ENC1, NKCC1, Ephrin-B receptor 1, HNF4-alpha, Cofilin, c-Myb, DMP4, Alpha/epsilon-sarcoglycan, AML1/TRPS1 fusion protein, NOX1, Vimentin, Osteonectin, SRGAP2, MyHC, PKC, CRMP4, MGP, PTPR-mu, G-protein gamma, Hedgehog, FA2H, SLC5A1, DFNA5, AKT(PKB), PKC-mu, NELL1, RILP (REST- interacting LIM domain protein), CAP2, Necdin, ECM2/SC1, MYH10, TRPC1, MYH14, TAZ, NAT-2, KCNQ1, TRPS1, BASP1, HNF4, STRBP, PRKD2, M-Ras, Decorin, JAM3 regulation of 2241 5.401E−07 8.554E−05 57 c-Maf, SLUG, Ephrin-B receptors, FLRT2, developmental Caspase-6, LZTS1, PEDF (serpinF1), Axin, FEZ1, process PLA2, GCR-alpha, ERR1, SPG20, Axin2, TIMP2, Plakophilin 2, FOG2, Galectin-1, CDC42EP5, Galpha(q)-specific peptide GPCRs, Kalirin, SHH, Ephrin-B receptor 2, DDAH1, HMGI/Y, LDB2, MAP-1B, Syndecan-2, GCR-beta, SLIT2, ERM proteins, Neuropilin-1, Ephrin-B receptor 1, HNF4- alpha, Cofilin, c-Myb, DMP4, AML1/TRPS1 fusion protein, Vimentin, SRGAP2, MyHC, PKC, CRMP4, MGP, PTPR-mu, Hedgehog, AKT(PKB), PKC-mu, NELL1, RILP (REST-interacting LIM domain protein), MYH10, MYH14, TAZ, TRPS1, BASP1, HNF4, PRKD2 regulation of 843 6.651E−07 1.016E−04 30 Ephrin-B receptors, FLRT2, Caspase-6, LZTS1, cell PEDF (serpinF1), Axin, FEZ1, GCR-alpha, SPG20, development Axin2, TIMP2, Galectin-1, Galpha(q)-specific peptide GPCRs, Kalirin, SHH, Ephrin-B receptor 2, MAP-1B, Syndecan-2, GCR-beta, SLIT2, Neuropilin-1, Vimentin, SRGAP2, PKC, CRMP4, Hedgehog, AKT(PKB), PKC-mu, RILP (REST- interacting LIM domain protein), TAZ regulation of 382 7.207E−07 1.063E−04 19 Ephrin-B receptors, LZTS1, FEZ1, GCR-alpha, neuron SPG20, Galectin-1, Galpha(q)-specific peptide projection GPCRs, Kalirin, Ephrin-B receptor 2, MAP-1B, development Syndecan-2, GCR-beta, SLIT2, Neuropilin-1, Vimentin, PKC, CRMP4, AKT(PKB), PKC-mu regulation of 125 7.570E−07 1.067E−04 11 SLUG, Neuropilin-2, Galpha(q)-specific peptide endothelial GPCRs, SLIT2, Neuropilin-1, PKC, PTPR-mu, cell migration AKT(PKB), PKC-mu, AMOTL1 (Jeap), PRKD2 cellular 1137 7.738E−07 1.067E−04 36 SLUG, Ephrin-B receptors, FLRT2, FEZ1, Tubulin component beta, Neuropilin-2, PLA2, SLIT3, morphogenesis MAP7(EMAP115), Galpha(q)-specific peptide GPCRs, Willin, UCHL1, Kalirin, SHH, Ephrin-B receptor 2, MAP-1B, Syndecan-2, SLIT2, ERM proteins, DZIP, Neuropilin-1, Ephrin-B receptor 1, Cofilin, SRGAP2, MyHC, PKC, CRMP4, PTPR- mu, Hedgehog, CAP2, Necdin, MYH10, TRPC1, MYH14, TAZ, BASP1 cell 1875 8.084E−07 1.080E−04 50 PAR2, c-Maf, QKI, SLUG, Alpha crystallin B, development Ephrin-B receptors, FLRT2, Axin, FEZ1, Tubulin beta, Neuropilin-2, PLA2, SLAC2-B, SLIT3, MAP7(EMAP115), Galpha(q)-specific peptide GPCRs, Willin, UCHL1, Kalirin, SHH, Ephrin-B receptor 2, LDB2, MAP-1B, Syndecan-2, SLIT2, ERM proteins, DZIP, Neuropilin-1, Ephrin-B receptor 1, Cofilin, DMP4, AML1/TRPS1 fusion protein, Vimentin, SRGAP2, MyHC, PKC, CRMP4, PTPR-mu, G-protein gamma, Hedgehog, FA2H, AKT(PKB), CAP2, Necdin, MYH10, TRPC1, MYH14, BASP1, STRBP, JAM3 tissue 1879 8.608E−07 1.115E−04 50 PAR2, c-Maf, QKI, SLUG, IQCJ-SCHIP1, BDH, development Axin, Neuropilin-2, PLA2, GCR-alpha, SLAC2-B, ERR1, SPG20, Axin2, Plakophilin 2, MAP7(EMAP115), FOG2, Calpain 5, Myosin Va, SCHIP1, Galpha(q)-specific peptide GPCRs, Willin, SHH, Decorin proteoglycan, DUOX2, LDB2, GCR- beta, SLIT2, Polycystin 2, ERM proteins, Neuropilin-1, NKCC1, Cofilin, DMP4, AML1/TRPS1 fusion protein, Vimentin, MyHC, PKC, MGP, Hedgehog, FA2H, AKT(PKB), RILP (REST-interacting LIM domain protein), MYH10, TRPS1, BASP1, STRBP, PRKD2, Decorin, JAM3 response to 1775 9.452E−07 1.189E−04 48 CCL28, CDS1, Ephrin-B receptors, FLRT2, BDH, external FEZ1, Tubulin beta, Neuropilin-2, PLA2, stimulus HSD11B2, SLIT3, Galpha(q)-specific peptide GPCRs, Caspase-5, SHH, CDP-diacylglycerol synthase, Ephrin-B receptor 2, Decorin proteoglycan, Cmtm3, MAP-1B, HOXB9, Syndecan-2, SLIT2, Polycystin 2, ERM proteins, Neuropilin-1, NKCC1, TRPA1, Ephrin-B receptor 1, AP-1 mu subunits, Cofilin, Vimentin, Osteonectin, SRGAP2, MyHC, PKC, CMTM4, CRMP4, MGP, PTPR-mu, G-protein gamma, Hedgehog, AKT(PKB), CAP2, MYH10, TRPC1, MYH14, STRBP, Decorin regulation of 634 9.767E−07 1.193E−04 25 PAR2, SLUG, CARD5, FLRT2, Neuropilin-2, cell migration PLA2, Plakophilin 2, Galpha(q)-specific peptide GPCRs, SHH, MSN (moesin), SLIT2, ERM proteins, Neuropilin-1, CRCM, Vimentin, SRGAP2, PKC, CRMP4, PTPR-mu, Hedgehog, AKT(PKB), PKC-mu, AMOTL1 (Jeap), PRKD2, JAM3 regulation of 157 1.082E−06 1.285E−04 12 SLUG, Neuropilin-2, Galpha(q)-specific peptide epithelial cell GPCRs, SLIT2, Neuropilin-1, CRCM, PKC, PTPR- migration mu, AKT(PKB), PKC-mu, AMOTL1 (Jeap), PRKD2 neuron 865 1.132E−06 1.308E−04 30 Ephrin-B receptors, FLRT2, FEZ1, Tubulin beta, projection Neuropilin-2, PLA2, SLIT3, Galpha(q)-specific development peptide GPCRs, UCHL1, Kalirin, SHH, Ephrin-B receptor 2, MAP-1B, Syndecan-2, SLIT2, ERM proteins, Neuropilin-1, Ephrin-B receptor 1, Cofilin, SRGAP2, MyHC, PKC, CRMP4, PTPR-mu, Hedgehog, CAP2, Necdin, MYH10, TRPC1, MYH14 negative 473 1.173E−06 1.320E−04 21 PAR2, SLUG, Alpha crystallin B, Axin, PLA2, regulation of Axin2, Galpha(q)-specific peptide GPCRs, SHH, transport Synaptotagmin, MAP-1B, Polycystin 2, HNF4- alpha, PKC, FAM3D, Hedgehog, PEA15, AKT(PKB), PKC-mu, TAZ, KCNQ1, HNF4 organ 3386 1.321E−06 1.449E−04 75 SNF2L1, PAR2, c-Maf, SLUG, Alpha crystallin B, development IQCJ-SCHIP1, AEBP1, Ephrin-B receptors, Caspase-6, SLC29A2, PEDF (serpinF1), BDH, Axin, Neuropilin-2, PLA2, LEC1, GCR-alpha, SLAC2-B, ERR1, SPG20, Axin2, SLIT3, Plakophilin 2, MAP7(EMAP115), FOG2, Calpain 5, Galectin-1, Myosin Va, SCHIP1, Galpha(q)-specific peptide GPCRs, Epsilon-sarcoglycan, SHH, DAZAP, Ephrin-B receptor 2, Decorin proteoglycan, CDX1, DUOX2, LDB2, HOXB9, GCR-beta, SLIT2, Polycystin 2, NNMT, Neuropilin-1, NKCC1, Ephrin-B receptor 1, HNF4- alpha, Cofilin, c-Myb, DMP4, Alpha/epsilon- sarcoglycan, AML1/TRPS1 fusion protein, Vimentin, Osteonectin, SRGAP2, MyHC, PKC, MGP, PTPR-mu, G-protein gamma, Hedgehog, FA2H, SLC5A1, DFNA5, AKT(PKB), MYH10, TAZ, NAT-2, KCNQ1, TRPS1, BASP1, HNF4, M- Ras, Decorin, JAM3 positive 8 1.903E−06 2.035E−04 4 SLUG, PKC, PKC-mu, PRKD2 regulation of endothelial cell chemotaxis cell migration 842 1.986E−06 2.042E−04 29 PAR2, SLUG, IQCJ-SCHIP1, Ephrin-B receptors, Tubulin beta, Tensin, Neuropilin-2, PLA2, Matriptase, SLIT3, SCHIP1, Galpha(q)-specific peptide GPCRs, SHH, MSN (moesin), HOXB9, SLIT2, ERM proteins, Neuropilin-1, Ephrin-B receptor 1, Cofilin, NOX1, SRGAP2, MyHC, PKC, Hedgehog, AMOTL1 (Jeap), Necdin, MYH10, JAM3 positive 65 2.006E−06 2.042E−04 8 SLUG, Neuropilin-2, Neuropilin-1, PKC, regulation of AKT(PKB), PKC-mu, AMOTL1 (Jeap), PRKD2 endothelial cell migration regulation of 669 2.556E−06 2.542E−04 25 PAR2, SLUG, CARD5, FLRT2, Neuropilin-2, cell motility PLA2, Plakophilin 2, Galpha(q)-specific peptide GPCRs, SHH, MSN (moesin), SLIT2, ERM proteins, Neuropilin-1, CRCM, Vimentin, SRGAP2, PKC, CRMP4, PTPR-mu, Hedgehog, AKT(PKB), PKC-mu, AMOTL1 (Jeap), PRKD2, JAM3 positive 115 2.703E−06 2.627E−04 10 PAR2, SLUG, PLA2, Galpha(q)-specific peptide regulation of GPCRs, SLIT2, Neuropilin-1, PKC, AKT(PKB), chemotaxis PKC-mu, PRKD2 plasma 143 2.867E−06 2.691E−04 11 MAP7(EMAP115), Myosin Va, Rab-31, Galpha(q)- membrane specific peptide GPCRs, Synaptotagmin, EHD3, organization MyHC, PKC, FA2H, AKT(PKB), MYH10 developmental 5978 2.895E−06 2.691E−04 113 SNF2L1, PAR2, c-Maf, QKI, RECK, SLUG, Alpha process crystallin B, IQCJ-SCHIP1, AEBP1, Ephrin-B receptors, FLRT2, Caspase-6, SLC29A2, LZTS1, PEDF (serpinF1), BDH, Axin, FEZ1, Tubulin beta, Neuropilin-2, DUOX1, PLA2, LEC1, GCR-alpha, MUPP1, SLAC2-B, ERR1, SPG20, Axin2, SLIT3, TIMP2, Plakophilin 2, MAP7(EMAP115), FOG2, Calpain 5, Galectin-1, Myosin Va, SCHIP1, Galpha(q)-specific peptide GPCRs, Epsilon- sarcoglycan, Willin, UCHL1, PAQR8, Kalirin, SHH, DAZAP, Ephrin-B receptor 2, Decorin proteoglycan, CDX1, HMGI/Y, FRAT2, DUOX2, TMPRSS2/ERG fusion protein, NAT-1, LDB2, MAP-1B, Chk2, HOXB9, Syndecan-2, GCR-beta, SLIT2, Glis2, Polycystin 2, NNMT, ERM proteins, DZIP, Neuropilin-1, ENC1, NKCC1, Ephrin-B receptor 1, SELENBP1, AP-1 mu subunits, HNF4- alpha, Cofilin, c-Myb, DMP4, Alpha/epsilon- sarcoglycan, AML1/TRPS1 fusion protein, NOX1, Vimentin, Osteonectin, SRGAP2, MyHC, PKC, CRMP4, MGP, PTPR-mu, G-protein gamma, Hedgehog, FA2H, SLC5A1, DFNA5, AKT(PKB), PKC-mu, NELL1, RILP (REST-interacting LIM domain protein), CAP2, Necdin, ECM2/SC1, MYH10, TRPC1, MYH14, TAZ, NAT-2, KCNQ1, TRPS1, BASP1, HNF4, STRBP, PRKD2, M-Ras, Decorin, JAM3 regulation of 1910 3.197E−06 2.881E−04 49 PAR2, SLUG, ASAP, Alpha crystallin B, CARD5, cellular Ephrin-B receptors, LZTS1, Axin, FEZ1, Stonin-1, component PLA2, GCR-alpha, SPG20, Axin2, SLIT3, organization Plakophilin 2, Galectin-1, CDC42EP5, Galpha(q)- specific peptide GPCRs, Kalirin, Synaptotagmin, Ephrin-B receptor 2, MAP-1B, Syndecan-2, GCR- beta, SLIT2, ERM proteins, Neuropilin-1, PRSS11 (HtrA1), Ephrin-B receptor 1, HNF4-alpha, Cofilin, c-Myb, NOX1, Vimentin, MyHC, PKC, CRMP4, G-protein gamma, AKT(PKB), PKC-mu, Necdin, MYH10, MYH14, TAZ, MLLT11, HNF4, PRKD2, JAM3 positive 92 3.234E−06 2.881E−04 9 SLUG, Neuropilin-2, Galpha(q)-specific peptide regulation of GPCRs, Neuropilin-1, PKC, AKT(PKB), PKC-mu, epithelial cell AMOTL1 (Jeap), PRKD2 migration vascular 19 3.646E−06 3.145E−04 5 Neuropilin-2, Neuropilin-1, PKC, PKC-mu, PRKD2 endothelial growth factor signaling pathway negative 119 3.686E−06 3.145E−04 10 SLUG, Alpha crystallin B, Axin, Axin2, MAP-1B, regulation of Polycystin 2, HNF4-alpha, PKC, TAZ, HNF4 intracellular transport

TABLE 12C Non-Overlapped Genes Maps pValue Nov Overlapping Genes Immune response_Inflammasome in inflammatory 2.075E−04 CARD5, IL-1RI, Caspase- response 5, Biglycan Neurophysiological process_EphB receptors in 2.327E−04 Syndecan-2, Ephrin-B dendritic spine morphogenesis and synaptogenesis receptors, Kalirin, Ephrin-B receptor 2 Cell adhesion_Tight junctions 2.600E−04 MUPP1, Claudin-3, ZO-3, CDX1 Development_TGF-beta-dependent induction of 6.742E−04 SLUG, Caldesmon, Cofilin, EMT via RhoA, PI3K and ILK. AKT(PKB) Development_Slit-Robo signaling 2.218E−03 SLIT2, Cofilin, SLIT3 Cell adhesion_Gap junctions 2.218E−03 Tubulin beta, PKC, ZO-3 Signal transduction_Erk Interactions: Inhibition of 3.188E−03 PKC, PEA15, AKT(PKB) Erk Development_VEGF-family signaling 5.435E−03 Neuropilin-1, Neuropilin-2, AKT(PKB) Cell adhesion_Ephrin signaling 7.056E−03 Ephrin-B receptors, Ephrin- B receptor 1, Kalirin Immune response_MIF - the neuroendocrine- 7.501E−03 PLA2, PKC, HMGI/Y macrophage connector Cell adhesion_ECM remodeling 1.052E−02 Syndecan-2, TIMP2, MSN (moesin) Development_WNT signaling pathway. Part 2 1.108E−02 SLUG, ENC1, Axin Cell adhesion_Chemokines and adhesion 1.135E−02 Syndecan-2, Cofilin, AKT(PKB), MSN (moesin) Development_FGF2-dependent induction of EMT 1.301E−02 IL-1RI, AKT(PKB) Cytoskeleton remodeling_ESR1 action on 1.564E−02 AKT(PKB), MSN (moesin) cytoskeleton remodeling and cell migration Cytoskeleton remodeling_Regulation of actin 1.703E−02 Cofilin, MyHC cytoskeleton by Rho GTPases Development_Regulation of epithelial-to- 1.840E−02 SLUG, Caldesmon, IL-1RI mesenchymal transition (EMT) Development_Glucocorticoid receptor signaling 1.847E−02 GCR-beta, GCR-alpha Transcription_Transcription regulation of aminoacid 1.997E−02 c-Maf, PKC metabolism Neolacto-series GSL Metabolism p.1 2.330E−02 FUT6, FUT2, FUT4 Neolacto-series GSL Metabolism p.1/Human 2.330E−02 FUT6, FUT2, FUT4 version Inhibition of neutrophil migration by proresolving 2.330E−02 IL-1RI, PKC, MSN lipid mediators in COPD (moesin) Proteolysis_Putative SUMO-1 pathway 2.642E−02 c-Myb, GCR-alpha Colorectal cancer (general schema) 2.815E−02 Ephrin-B receptors, SHH Transport_Aldosterone-mediated regulation of ENaC 2.815E−02 GCR-alpha, AKT(PKB) sodium transport Regulation of lipid metabolism_FXR-dependent 2.992E−02 PXR, HNF4-alpha negative-feedback regulation of bile acids concentration Cytoskeleton remodeling_Reverse signaling by 2.992E−02 Ephrin-B receptors, Axin ephrin B Linoleic acid/Rodent version 2.992E−02 PLA2G12, CYP2J2 Putative pathways for stimulation of fat cell 3.174E−02 GCR-alpha, AKT(PKB) differentiation by Bisphenol A Development_Transcription regulation of granulocyte 3.174E−02 c-Myb, PKC development Apoptosis and survival_Caspase cascade 3.360E−02 Caspase-6, AKT(PKB) Cytoskeleton remodeling_Keratin filaments 3.942E−02 Tubulin beta, Plakophilin 2 Development_MAG-dependent inhibition of neurite 4.144E−02 Cofilin, MyHC outgrowth Regulation of metabolism_Bile acids regulation of 4.144E−02 HNF4-alpha, AKT(PKB) glucose and lipid metabolism via FXR Cell adhesion_Cell-matrix glycoconjugates 4.350E−02 Galectin-4, TIMP2

Interestingly, in comparing EMT to ΔPC1.EMT, the gene with the greatest weight change was CD24 (FIG. 3I), previously reported as a metastasis-associated gene with high expression linked to worse survival in bladder cancer (Smith. S. C. et al. Cancer Res. 2006 66(4):1917-22). CD24 was also reported as a marker of colon cancer stem cells (CSC) whose properties are thought to contribute to “metastatic traits” and therapeutic resistance (Oskarsson, T., Cell Stem Cell. 2014 14(3):306-21; Ashley, N. et al. Cancer Res. 2013 73(18):5798-809). Thus, ΔPC1. EMT may capture the epithelial CSC features in CRC, which is consistent with a recent report demonstrating that in breast cancer, CDH1 and CD24 were highly enriched in the epithelial CSCs characterized by expression of the CSC marker ALDH1, while their expression was down-regulated in the mesenchymal CSCs (CD44⁺CD24⁻) (Liu, S. et al. Stem Cell Reports. 2013 2(1):78-91). ERBR3 was also identified as one of the genes whose contribution was increased in ΔPC1.EMT (FIG. 3I) and was amplified, overexpressed, or mutated in human cancers (Guinney, J. et al. Clin Cancer Res. 2014 20(1):265-72). In agreement with ERBB3 activation, ΔPC1.EMT, but not EMT, was associated with activation of the RAS/MAPK pathway, evidenced by its positive correlation with various RAS and BRAF scores (Table 13 and FIG. 20). Since abnormal activation of the RAS/MAPK pathway is thought to contribute to the resistance of anti-EGFR treatments (Guinney, J. et al. Clin Cancer Res. 2014 20(1):265-72), ΔPC1.EMT-associated poor prognosis might, in part, result from RAS/MAPK-mediated drug resistance in epithelial-like CRC.

TABLE 13 Correlations-Moffitt468 d_meta APC_ TP53 KRAS EMT_Score PC1_Score d_meta 1 0.09487 0.09409 0.16646 0.12244 0.26862 0.0402 0.0419 0.0003 0.008 <.0001 APC_ 0.09487 1 0.13219 0.25802 −0.09383 −0.20689 0.0402 0.0042 <.0001 0.0425 <.0001 TP53 0.09409 0.13219 1 −0.08521 −0.04201 −0.06056 0.0419 0.0042 0.0655 0.3645 0.1909 KRAS 0.16646 0.25802 −0.08521 1 −0.14592 −0.09788 0.0003 <.0001 0.0655 0.0015 0.0343 EMT_Score 0.12244 −0.09383 −0.04201 −0.14592 1 0.9 0.008 0.0425 0.3645 0.0015 <.0001 PC1_Score 0.26862 −0.20689 −0.06056 −0.09788 0.9 1 <.0001 <.0001 0.1909 0.0343 <.0001 ΔPC1.EMT 0.3845 −0.26895 −0.04665 0.0195 0.38 0.74 <.0001 <.0001 0.314 0.6739 <.0001 <.0001 RAS_Merck_ −0.09597 −0.15514 −0.0884 0.1156 −0.10964 0.01908 0.038 0.0008 0.056 0.123 0.0177 0.6805 RAS_Astra 0.08945 −0.10735 −0.15836 0.39651 −0.01907 0.14859 0.0531 0.0202 0.0006 <.0001 0.6807 0.0013 BRAF_ −0.10847 −0.39096 −0.08739 −0.29806 −0.04731 0.08427 0.0189 <.0001 0.0589 <.0001 0.3071 0.0685 msi_high −0.1927 −0.33209 −0.25961 −0.15516 −0.14551 −0.02419 <.0001 <.0001 <.0001 0.0008 0.0016 0.6016 ΔPC1.EMT RAS_Merck_ RAS_Astra BRAF_ msi_high d_meta 0.3845 −0.09597 0.08945 −0.10847 −0.1927 <.0001 0.038 0.0531 0.0189 <.0001 APC_ −0.26895 −0.15514 −0.10735 −0.39096 −0.33209 <.0001 0.0008 0.0202 <.0001 <.0001 TP53 −0.04665 −0.0884 −0.15836 −0.08739 −0.25961 0.314 0.056 0.0006 0.0589 <.0001 KRAS 0.0195 0.1156 0.39651 −0.29806 −0.15516 0.6739 0.123 <.0001 <.0001 0.0008 EMT_Score 0.38 −0.10964 −0.01907 −0.04731 −0.14551 <.0001 0.0177 0.6807 0.3071 0.0016 PC1_Score 0.74 0.01908 0.14859 0.08427 −0.02419 <.0001 0.6805 0.0013 0.0685 0.6016 ΔPC1.EMT 1 0.15645 0.33994 0.22097 0.14016 0.0007 <.0001 <.0001 0.0024 RAS_Merck_ 0.15645 1 0.48973 0.28309 0.33283 0.0007 <.0001 <.0001 <.0001 RAS_Astra 0.33994 0.48973 1 0.32409 0.31115 <.0001 <.0001 <.0001 <.0001 BRAF_ 0.22097 0.28309 0.32409 1 0.5626 <.0001 <.0001 <.0001 <.0001 msi_high 0.14016 0.33283 0.31115 0.5626 1 0.0024 <.0001 <.0001 <.0001

A list of ten up-regulated and ten down-regulated genes (Tables 14A and 14B) were identified whose expression was correlated with ΔPC1.EMT in a linear model on the five datasets plus the TCGA CRC dataset (Cancer Genome Atlas Network. Nature. 2012 487(7407):330-7), and interestingly, about half of the identified genes are overlapped with the PC1 and EMT signature genes (especially PC1 down genes), suggesting these genes may maintain similar contributions between ΔPC1.EMT and parent scores. The great majority of identified genes have been reported to have biological functions related to epithelial or mesenchymal biology or to metastasis. For instance, CD109 (top up-regulated gene) has recently identified by proteomic analyses as a metastasis-associated protein marker (Karhemo, P. R. et al. J Proteomics. 2012 77:87-100), and CD109 was highly expressed in ALDH1-characterized epithelial sarcoma CSCs (Emori, M. et al. PLoS One. 2013 8(12):e84187). CDX/and CDX2 (top two down-regulated genes) were reported as putative tumor suppressor genes whose expression was epigenetically repressed in CRC, and reduced expression of CDX1 inhibited CSC stem cell differentiation and thus promoted CSC renewal (Ashley, N. et al. Cancer Res. 2013 73(18):5798-809). In support of this, HCT116, an epithelial, MSI CRC cell line that lacks expression of CDX1 was recently classified as a colon CSC cell line (Sadanandam, A. et al. Nat Med. 2013 19(5):619-25). In addition, reduced expression of EPHB2 was associated with metastasis (Yu, G. et al. J Cancer Res Clin Oncol. 2011 137(1):73-80) while its overexpression induced EMT (Gao, Q. et al. Hum Pathol. 2014 45(2):372-81). Another down-regulated gene, MYB, is a cell cycle gene, and its ectopic expression was reported to contribute to cell migration and invasion but to also prevent metastasis (Knopfova, L. et al. Mol Cancer. 2012 11:15). It is noteworthy that inhibition of cell proliferation is thought to be necessary in the tumor dormancy step of metastasis (Giancotti, F. G. Cell. 2013 155(4):750-64). Thus, identification EPHB2 and MYB as ΔPC1.EMT-correlated down-regulated genes further supports the notion of non-EMT contributions to metastasis.

TABLE 14A Top ten genes positively correlated with ΔPC1.EMT Gene Entrez- Sum t Symbol ID S num p p value p adj statistics CD109 135228 869.79 5 0.00 0.00 75.05 AHNAK2 113146 837.41 6 0.00 0.00 79.32 GAS1 2619 806.50 6 0.00 0.00 76.57 PRKCDBP 112464 806.43 6 0.00 0.00 77.90 MEIS2 4212 779.02 6 0.00 0.00 77.16 NXN 64359 772.64 5 0.00 0.00 70.33 GFPT2 9945 727.95 6 0.00 0.00 72.26 Overlapped UP PMP22 5376 711.36 6 0.00 0.00 73.46 EMT Up WWTR1 25937 692.29 6 0.00 0.00 72.07 PC1 Up PTRF 284119 688.52 6 0.00 0.00 71.22 Overlapped Up

TABLE 14B Top ten genes negatively correlated with ΔPC1.EMT Gene Entrez- Sum t Symbol ID S num p p value p adj statistics CDX1 1044 860.61 6 0.00 0.00 −80.16 PC1 Down CDX2 1045 845.27 6 0.00 0.00 −79.41 C10orf99 387695 767.33 5 0.00 0.00 −67.82 PC1 Down DDC 1644 752.19 6 0.00 0.00 −73.57 GPA33 10223 726.29 6 0.00 0.00 −72.98 PC1 Down FAM84A 151354 720.55 5 0.00 0.00 −67.43 Overlapped Down NR1I2 8856 697.98 6 0.00 0.00 −70.24 PC1 Down MYB 4602 630.56 6 0.00 0.00 −68.13 PC1 Down C2orf89 129293 616.89 5 0.00 0.00 −60.62 EPHB2 2048 597.82 6 0.00 0.00 −66.42 PC1 Down

Gene set enrichment analysis identified a variety of biological processes correlated with ΔPC1.EMT, including negatively correlated mitochondrial metabolism (Tables 27 to 42), a trait of epithelial stem cells. It is noteworthy that metastasis suppressor gene KISS1 was recently reported to promote normal mitochondrial metabolism, an anti-metastasis mechanism (Favre, C., et al. Oncogene. 2010 29(27):3964-76). Finally, the association of the ΔPC1.EMT score with an expanded set of other known prognostic signatures was tested on the five datasets in a univariate analysis. Results showed that ΔPC1.EMT was the signature that, overall, had the highest significant prognostic value for OS and RFS across all the datasets tested (FIGS. 21A to 21H). It is of interest to mention that ΔPC1.EMT showed a relatively high correlation with the OncotypeDX™ colon signature (Genomic Health, Inc.) (Table 15), which had exploited cell proliferation as a potential prognostic marker, and GH score was found to be negatively correlated with cell cycle genes such as MYBL2 (O'Connell, M. J. et al. J Clin Oncol. 2010 28(25):3937-44), a family member of MYB as mentioned earlier. Taken together, the negative association of ΔPC1.EMT with growth properties such as mitochondrial metabolism and cell proliferation may be non-EMT processes captured by the score.

TABLE 15 Correlations between ΔPC1.EMT and OncotypeDX colon signature (GH) scores Datasets Pearson's Corr. P.value N samples PETACC3 0.36 95% CI (0.29-0.42) <2.2e−16 N = 750 ALMAC 0.31 95% CI (0.21-0.40) 2.48e−09 N = 357 LNCC 0.68 95% CI (0.63-0.72) <2.2e−16 N = 564 GSE14333 0.69 95% CI (0.62-0.74) <2.2e−16 N = 288 GEO41258 0.78 95% CI (0.72-0.83) <2.2e−16 N = 184

In conclusion, while EMT appears to be a dominant program in CRC, ΔPC1.EMT is far more predictive of CRC outcome (metastasis and survival) than its parent PC1 or EMT scores. Moreover, it is the “best in class” when compared to a variety of other known prognostic signatures. The subtraction of EMT from PC1 (ΔPC1.EMT) increases its bias in detecting non-EMT biology, including epithelial CSCs, thereby improving its potential to portend metastasis and providing new targets for therapy of distant disease. These observations support the hypothesis that both epithelial and mesenchymal cell phenotypes cooperate to produce metastasis (Tsuji, T et al. Cancer Res. 2009 69(18):7135-9; Nieto, M. A. Science. 2013 342(6159):1234850).

Methods

Moffitt468 and additional five independent datasets, including PTEACC31, ALMAC2, LNCC3, GEO412584 and GSE143335 (Budinska, E. et al. J Pathol. 2013 231(1):63-76; Kennedy, R. D. et al. J Clin Oncol. 2011 29(35):4620-6; Marisa, L. et al. PLoS Med. 2013 10(5):e1001453; Sheffer, M. et al. Proc Natl Acad Sci USA. 2009 106(17):7131-6; Jorissen, R. N. et al. Clin Cancer Res. 2009 15(24):7642-7651) were tested. Probe intensities were preprocessed using RMA. PC1 and EMT scores were calculated as previously described (Loboda, A. et al. BMC Med Genomics. 2011 4:9). Briefly, for each of the datasets, a score was computed for each of the 4 signatures (EMT.UP.score, EMT.DOWN.score, PC1.UP.score and PC1.DOWN.score) as the arithmetic mean of all probesets corresponding to gene symbols present in the corresponding gene signature. EMT and PC1 scores were then obtained as follows:

EMT.scorc=EMT.UP.score−EMT.DOWN.score

PC1.score=PC1.UP.score−PC1.DOWN.score

The ΔPC1.EMT score was computed as follows: ΔPC1.EMT.score=PC1.score−EMT.score Scores were standardized by subtracting the score median and dividing by the score IQR.

Pearson's product moment correlation coefficient was used to quantify the association between the scores, MSI status, and mutation status for various genes. Pathways analyses of the non-overlapped genes of PC1 and EMT signatures by GO Process were performed using the MetaCore package. A P-values cut-off of 0.05 resulted in 35 significant dysregulated pathways.

The association of gene expression with the ΔPC1.EMT.score within each of the datasets was tested by a linear regression model with the score as the explanatory variable using the “limma” R package (version 3.16.3), adjusting standard errors estimates by an empirical Bayes approach. P-values were combined across datasets using Fisher's method (MADAM R package version 1.2.2). A Bonferroni correction was applied to control for false positive results introduced by multiple testing.

Genes showing an adjusted P-value <0.00001 were split in two groups: those positively (N=2,983) and those negatively (N=2,221) correlated with the ΔPC1.EMT score. The functional tool DAVID (http://david.abcc.ncifcrf.gov/) was employed to identify annotation terms enriched within each of the groups. The 15,896 genes measured in all 5 datasets were used as background. The scores were computed from 10 signatures (RAS Merck (Loboda, A. et al. BMC Med Genomics. 2010 3:26) RAS Astrazeneca (Dry, J. R. et al. Cancer Res. 2010 70(6):2264-73), OncotypeDX colon (O'Connell, M. J. et al. J Clin Oncol. 2010 28(25):3937-44), Veridex (Jiang, Y. et al. J Mol Diagn. 2008 10(4):346-5), MD Anderson (Oh, S. C. et al. Gut. 2012 61(9):1291-8), Decorin (Farmer, P. et al. Nat Med. 2009 15(1):68-74), MED12 (Huang, S. et al. Cell. 2012 151(5):937-50), BRAF score (Popovici, V. et al. J Clin Oncol. 2012 30(12):1288-95) and ALM (Kennedy, R. D. et al. J Clin Oncol. 2011 29(35):4620-6) as described in the original study. Cox proportional hazards regression models was used in the R package “survival” (version 2.37-7) to assess association of tumor scores with Overall Survival (OS), Relapse-free survival (RFS) and Survival after Relapse (SAR).

In order to characterize the three signatures (PC1, EMT and ΔPC1.EMT), the average contribution of each gene was estimated to each of the signatures across five data sets. For each data set, a contribution was first calculated for each probe set to the PC1 and EMT signatures, respectively. The contribution was proportional to the average expression level of the probe set and inversely proportional to the number of probe sets included in the signature for the microarray platform used for the data set. Then, gene-wise contributions were estimated to each signature by summing the contributions for all probe sets corresponding to the same gene. The contributions to the ΔPC1.EMT signature were obtained as the difference between the contributions to the PC1 and the EMT signatures. Finally, a weighted average of the contributions was computed across all five data sets to obtain final estimates of the gene contributions to the three signatures. The weight for a data set was inversely proportional to the Euclidean norm of the vector of gene contributions to the PC1 and EMT signatures in the data set. A linear contrast was used to test for a trend in gene expression score with increasing stage of primary disease to distant metastasis, using PROC GLM (SAS, version 9.2).

Example 2

Table 16 summarizes the main features of the datasets used in this Example.

TABLE 16 Study Datasets Institution RNA Dataset (Article) Accession n Stage Treatment Platform material Survival PETACC3 BCF, E-MTAB-990 752 2, 3 5-FU/FA Almac FFPE OS, RFS, Lausanne FOLFIRI SAR (Budinska E, et. al.) ALMAC Queen's E-MTAB-863 359 2 Untreated^(a) Almac FFPE OS, RFS University E-MTAB-864 Belfast (Kennedy R D, et. al.) French Ligue E-GEOD-39582 566 1-4 Treated HGU133plus2 Frozen RFS Nationale Untreated Controle Cancer, Paris (Marisa L, et. al.) GEO41258 Weizmann E-GEOD-41258 186 1-4 Unknown HGU133a Frozen OS, RFS Institute of Science, Rehovot (Sheffer M, et. al.) GSE14333 Ludwig GSE14333 290 A-D Unknown^(b) HGU133plus2 Frozen RFS Institute for Cancer Research, Melbourne (Jorissen R N, et. al.) TCGA TCGA (The TCGA portal 385 1-4 Unknown RNA-seq Frozen poor Cancer Genome Atlas Network) ^(a)no preoperative or postoperative cancer therapy within 1 year of surgery (although therapy given after recurrence was acceptable) ^(b)standard adjuvant chemotherapy (either single agent 5-fluouracil/capocitabine or 5-fluouracil and oxaliplatin) or postoperative concurrent chemoradiotherapy (50.4 Gy in 28 fractions with concurrent 5-fluorouracil)

Correlation of PC1.EMT, PC1 and EMT Scores with Other Known Prognostic Signatures

As discussed, the association of PC1.EMT with other known prognostic signatures was tested. Specifically, PC1.EMT was compared with Oncotype DX, Mammaprint, RAS Merck, RAS Astrazeneca, Genomic Health colon signature [O'Connell M J, et al. (2010). J Clin Oncol.; 28:3937-44], Veradex [Jiang Y, et al. (2008). J Mol Diagn.; 10:346-54], MD Anderson signature [Oh S C, et al. (2012). Gut. 61:1291-8], Decorin signature [Farmer P, et al. (2009). Nat Med. 15:68-74], EMT signature [Loboda A, et al. (2011). BMC Med Genomics. 4:9], MED12 signature [Huang S1, et al. (2012). Cell. 151:937-50], BRAF signature [Popovici V, et al. (2012). J Clin Oncol. 30:1288-95], Coppola2011 signature [Coppola D, et al. (2011). Am J Pathol. 179:37-45], Peng2010 signature [Peng J, et al. (2010), Int J Colorectal Dis. 25:1277-85], Schetter2009 signature [Schetter A J, et al. (2009). Clin Cancer Res. 15:5878-87], Staub2009 signature [Staub E1, et al. (2009). J Mol Med (Berl). 87:633-44], and ALM signature [Kennedy RD1, et al. (2011). J Clin Oncol. 35:4620-6]. The comparison was performed in all available dataset.

FIGS. 6A to 6F are pairs plots of the signature scores in PETACC (FIG. 6A), ALAMC (FIG. 6B), French (FIG. 6C), GSE14333 (FIG. 6D), GEO41258 (FIG. 6E), and TCGA (FIG. 6F) datasets. FIGS. 7A to 7F show gene clusters for PETACC (FIG. 7A), ALAMC (FIG. 7B), French (FIG. 7C), GSE14333 (FIG. 7D), GEO41258 (FIG. 7E), and TCGA (FIG. 7F) datasets.

Based on the clustering there seem to be three stable groups of signatures: Group1: Oncotype TX, Mammaprint Coppola and Veridex Group2: Decorin, EMT MED12, Peng and Genomic Health; and Group3: BRAF, MDA and RAS.Merck. Some other elements moved between Group 2 and Group 3, including PC1.EMT.

Tables 17 to 24 show the correlation between prognostic signatures and OS/RFS.

TABLE 17 Univariate Cox Proportional Hazard Regression model for OS in Stage 2 and 3 - PETACC dataset HR (95% CI) P-value n PC1.EMT 1.56 (1.32-1.84) 1.16e−07 752 OncotypeTx 0.63 (0.53-0.75) 4.36e−07 752 MammaPrint 0.75 (0.62-0.90) 2.48e−03 752 RAS.Merck 0.98 (0.81-1.18)  8.4e−01 752 RAS.Astra 1.01 (0.84-1.21)  9.1e−01 752 GH 1.37 (1.15-1.63) 5.32e−04 752 Veridex 1.12 (0.93-1.35) 2.19e−01 752 MDA 1.36 (1.14-1.64) 8.08e−04 752 DCN 1.16 (0.96-1.40) 1.14e−01 752 EMT 1.22 (1.03-1.44) 2.01e−02 752 MED12 1.29 (1.06-1.58) 1.12e−02 752 BRAF 1.49 (1.24-1.80) 1.93e−05 752 Coppola 1.06 (0.89-1.26) 5.24e−01 752 Peng 1.16 (0.97-1.39) 9.39e−02 752 Schetter 1.32 (1.14-1.54) 2.77e−04 752 Staub 0.81 (0.67-0.98)  3.2e−02 752 ALM 1.18 (0.99-1.42) 7.03e−02 752

TABLE 18 Univariate Cox Proportional Hazard Regression model for RFS in Stage 2 and 3 - PETACC dataset HR (95% CI) P-value n PC1.EMT 1.47 (1.28-1.69) 8.98e−08 752 OncotypeTx 0.69 (0.59-0.80) 1.54e−06 752 MammaPrint 0.75 (0.63-0.88) 4.53e−04 752 RAS.Merck 0.97 (0.83-1.14)  7.3e−01 752 RAS.Astra 1.03 (0.88-1.19) 7.46e−01 752 GH 1.33 (1.14-1.55) 2.42e−04 752 Veridex 1.19 (1.01-1.39) 3.48e−02 752 MDA 1.15 (0.98-1.34) 8.54e−02 752 DCN 1.20 (1.02-1.41)  2.5e−02 752 EMT 1.22 (1.06-1.41) 5.95e−03 752 MED12 1.31 (1.10-1.55) 1.92e−03 752 BRAF 1.18 (1.00-1.38) 5.03e−02 752 Coppola 1.12 (0.96-1.30) 1.53e−01 752 Peng 1.19 (1.02-1.38) 2.67e−02 752 Schetter 1.27 (1.12-1.45) 2.11e−04 752 Staub 0.84 (0.71-0.98)   3e−02 752 ALM 1.18 (1.01-1.37) 4.12e−02 752

TABLE 19 Univariate Cox Proportional Hazard Regression model for OS in ALMAC dataset HR (95% CI) P-value n PC1.EMT 1.38 (1.14-1.68) 1.22e−03 359 OncotypeTx 0.94 (0.76-1.17) 5.72e−01 359 MammaPrint 1.17 (0.91-1.49) 2.15e−01 359 RAS.Merck 1.10 (0.87-1.38) 4.32e−01 359 RAS.Astra 1.42 (1.14-1.78) 2.03e−03 359 GH 1.40 (1.12-1.75) 3.01e−03 359 Veridex 1.19 (0.96-1.48) 1.08e−01 359 MDA 1.30 (1.02-1.66) 3.24e−02 359 DCN 1.44 (1.13-1.84) 3.22e−03 359 EMT 1.05 (0.85-1.30) 6.38e−01 359 MED12 1.24 (0.98-1.56) 7.51e−02 359 BRAF 1.12 (0.87-1.45) 3.75e−01 359 Coppola 1.00 (0.80-1.26) 9.83e−01 359 Peng 1.04 (0.85-1.26) 7.13e−01 359 Schetter 1.26 (1.08-1.46) 3.01e−03 359 Staub 1.20 (0.94-1.52) 1.43e−01 359 ALM 3.74 (2.92-4.79)   0e+00 359

TABLE 20 Univariate Cox Proportional Hazard Regression model for RFS in ALMAC dataset HR (95% CI) P-value n PC1.EMT 1.31 (1.08-1.59) 6.41e−03 359 OncotypeTx 1.03 (0.83-1.28) 7.86e−01 359 MammaPrint 1.13 (0.88-1.43) 3.38e−01 359 RAS.Merck 1.07 (0.86-1.35) 5.33e−01 359 RAS.Astra 1.40 (1.12-1.75) 2.95e−03 359 GH 1.40 (1.13-1.75) 2.64e−03 359 Veridex 1.12 (0.90-1.38) 3.16e−01 359 MDA 1.17 (0.92-1.48) 1.99e−01 359 DCN 1.49 (1.17-1.90) 1.14e−03 359 EMT 1.09 (0.88-1.35) 4.15e−01 359 MED12 1.29 (1.02-1.62) 3.04e−02 359 BRAF 1.02 (0.79-1.31) 8.78e−01 359 Coppola 1.07 (0.85-1.36) 5.47e−01 359 Peng 1.06 (0.87-1.29) 5.93e−01 359 Schetter 1.27 (1.08-1.49) 3.16e−03 359 Staub 1.21 (0.96-1.53) 1.05e−01 359 ALM 3.79 (2.98-4.82)   0e+00 359

TABLE 21 Univariate Cox Proportional Hazard Regression model for RFS in French dataset HR (95% CI) P-value n PC1.EMT 1.37 (1.16-1.62) 2.06e−04 557 OncotypeTx 0.75 (0.64-0.88)  5.9e−04 557 MammaPrint 0.81 (0.67-0.97) 2.09e−02 557 RAS.Merck 1.01 (0.83-1.24) 8.98e−01 557 RAS.Astra 1.28 (1.05-1.56) 1.36e−02 557 GH 1.53 (1.27-1.86) 9.53e−06 557 Veridex 1.05 (0.87-1.27) 6.24e−01 557 MDA 1.12 (0.91-1.37) 2.92e−01 557 DCN 1.42 (1.14-1.77) 2.07e−03 557 EMT 1.34 (1.12-1.61) 1.71e−03 557 MED12 1.53 (1.20-1.95) 7.04e−04 557 BRAF 1.10 (0.90-1.34) 3.52e−01 557 Coppola 1.05 (0.84-1.30)  6.7e−01 557 Peng 1.31 (1.08-1.59) 5.48e−03 557 Schetter 1.19 (0.99-1.45) 7.06e−02 557 Staub 1.27 (1.04-1.55) 1.99e−02 557 ALM 1.53 (1.26-1.86) 1.99e−05 557

TABLE 22 Univariate Cox Proportional Hazard Regression model for RFS in GSE14333 dataset HR (95% CI) P-value n PC1.EMT 2.66 (1.89-3.73) 1.53e−08 226 OncotypeTx 0.79 (0.61-1.04) 9.06e−02 226 MammaPrint 0.95 (0.65-1.37) 7.64e−01 226 RAS.Merck 1.54 (0.99-2.39) 5.59e−02 226 RAS.Astra 1.44 (0.96-2.15) 7.86e−02 226 GH 1.98 (1.38-2.84) 2.02e−04 226 Veridex 1.15 (0.78-1.69) 4.75e−01 226 MDA 2.02 (1.40-2.91) 1.87e−04 226 DCN 2.00 (1.35-2.95) 4.88e−04 226 EMT 1.51 (1.07-2.12) 1.79e−02 226 MED12 2.32 (1.49-3.59) 1.71e−04 226 BRAF 1.70 (1.21-2.39) 2.32e−03 226 Coppola 0.88 (0.60-1.30) 5.21e−01 226 Peng 1.08 (0.79-1.49) 6.35e−01 226 Schetter 0.88 (0.61-1.26) 4.74e−01 226 Staub 1.15 (0.79-1.70) 4.64e−01 226 ALM 1.80 (1.29-2.53) 5.99e−04 226

TABLE 23 Univariate Cox Proportional Hazard Regression model for OS in GEO41258 dataset HR (95% CI) P-value n PC1.EMT 1.44 (1.11-1.85) 5.66e−03 185 OncotypeTx 0.76 (0.58-1.01) 5.82e−02 185 MammaPrint 0.88 (0.68-1.13) 3.06e−01 185 RAS.Merck 1.11 (0.87-1.41) 4.02e−01 185 RAS.Astra 1.18 (0.97-1.44) 1.06e−01 185 GH 1.31 (1.01-1.69) 4.06e−02 185 Veridex 1.09 (0.84-1.41) 5.09e−01 185 MDA 1.04 (0.76-1.43) 7.88e−01 185 DCN 1.19 (0.91-1.56) 2.12e−01 185 EMT 1.23 (0.94-1.62) 1.32e−01 185 MED12 1.54 (1.12-2.13) 8.07e−03 185 BRAF 1.04 (0.76-1.41) 8.09e−01 185 Coppola 1.27 (0.96-1.70) 9.66e−02 185 Peng 0.96 (0.75-1.23) 7.67e−01 185 Schetter 1.02 (0.78-1.33) 9.04e−01 185 Staub 0.83 (0.63-1.10) 1.94e−01 185 ALM 1.29 (0.99-1.69) 6.14e−02 185

TABLE 24 Univariate Cox Proportional Hazard Regression model for RFS in GEO41258 dataset HR (95% CI) P-value n PC1.EMT 1.76 (1.17-2.67) 7.06e−03 109 OncotypeTx 0.77 (0.50-1.18) 2.27e−01 109 MammaPrint 1.09 (0.72-1.65) 6.85e−01 109 RAS.Merck 1.08 (0.71-1.64) 7.33e−01 109 RAS.Astra 1.72 (1.13-2.63) 1.23e−02 109 GH 1.52 (1.00-2.32) 5.16e−02 109 Veridex 1.11 (0.70-1.75) 6.48e−01 109 MDA 1.30 (0.78-2.16) 3.07e−01 109 DCN 1.44 (0.92-2.25) 1.09e−01 109 EMT 1.50 (0.95-2.35) 7.91e−02 109 MED12 1.54 (0.92-2.56) 9.72e−02 109 BRAF 1.22 (0.75-1.98) 4.24e−01 109 Coppola 1.10 (0.72-1.68) 6.55e−01 109 Peng 1.17 (0.81-1.70) 3.99e−01 109 Schetter 1.09 (0.69-1.74) 7.15e−01 109 Staub 1.05 (0.67-1.67) 8.21e−01 109 ALM 2.39 (1.48-3.87) 3.75e−04 109

Comparison of PC1.EMT with APC Mutations

PC1.EMT was compared with APC mutation status. For this analysis TCGA dataset was used. FIGS. 8A to 8B are boxplots with PC1.EMT score split by APC mutation status (WT VS all detrimental mutations or WT VS specific mutation type (known COSMIC)). No clear correlation was seen between PC1.EMT score and APC mutation status. However, some specific APC mutations showed lower score compared with WT.

Genes Correlating with PC1.EMT Signature Score

In order to have clues concerning the biological functions captured by the PC1.EMT score, we identified genes which expression correlates with the score using a linear model including only the PC1.EMT score. We used a meta-analytic method (Fisher) to merge the results across all 6 datasets. Tables 25 and 26 show the top 10 most consistent positive and negative correlating genes.

TABLE 25 Top 10 genes positively correlating with PC1.EMT score Entrez- Sum t Gene.Symbol ID S num. p p. value p. adj statistics CD109 135228 869.79 5 0.00 0.00 75.05 AHNAK2 113146 837.41 6 0.00 0.00 79.32 GAS1 2619 806.50 6 0.00 0.00 76.57 PRKCDBP 112464 806.43 6 0.00 0.00 77.90 MEIS2 4212 779.02 6 0.00 0.00 77.16 NXN 64359 772.64 5 0.00 0.00 70.33 GFPT2 9945 727.95 6 0.00 0.00 72.26 PMP22 5376 711.36 6 0.00 0.00 73.46 WWTR1 25937 692.29 6 0.00 0.00 72.07 PTRF 284119 688.52 6 0.00 0.00 71.22

TABLE 26 Top 10 genes negatively correlating with PC1.EMT score Entrez- Sum t Gene.Symbol ID S num. p p. value p. adj statistics CDX1 1044 860.61 6 0.00 0.00 −80.16 CDX2 1045 845.27 6 0.00 0.00 −79.41 C10orf99 387695 767.33 5 0.00 0.00 −67.82 DDC 1644 752.19 6 0.00 0.00 −73.57 GPA33 10223 726.29 6 0.00 0.00 −72.98 FAM84A 151354 720.55 5 0.00 0.00 −67.43 NR.1I2 8856 697.98 6 0.00 0.00 −70.24 MYB 4602 630.56 6 0.00 0.00 −68.13 C2orf89 129293 616.89 5 0.00 0.00 −60.62 EPHB2 2048 597.82 6 0.00 0.00 −66.42

FIG. 9A to 9D are plots of AHNAK2 gene log intensities as a function of PC1.EMT score intensities in PETACC (FIG. 9A), French (FIG. 9B), GEO41258 (FIG. 9C), and ALAMC (FIG. 9D) datasets. Example of gene positively correlating with the score. FIG. 10A to 10D are plots of CDX11 gene log intensities as a function of PC1.EMT score intensities in PETACC (FIG. 10A), French (FIG. 10B), GEO41258 (FIG. 10C), and ALAMC (FIG. 10D) datasets. Example of gene negatively correlating with the score

In order to interpret the list of genes found to be correlating with PC1.EMT score, gene enrichment analysis (GSEA) was performed using DAVID bioinformatics DB. Genes were split in two groups: list of genes found to be significantly positively correlated with PC1.EMT at an adjusted p value<0.05 (N=2351) or negatively correlated (N=1339). The two lists were submitted to the DAVID webpage and compared to the total number of analyzed gene (N=22946). Tables 27 and 28 show the top clusters of terms found to be enriched when using Functional annotation clustering tool:

TABLE 27 Top 10 Annotation clusters found enriched among genes positively correlating with PC1.EMT score (DAVID DB) Fold Category Term Count % PValue Enrichment FDR Annotation Cluster 1 Enrichment Score: 45.78660446516211 SP_PIR_KEYWORDS signal 704 30.1 0.0000 1.8 0.0000 UP_SEQ_FEATURE signal peptide 704 30.1 0.0000 1.8 0.0000 SP_PIR_KEYWORDS glycoprotein 820 35.1 0.0000 1.6 0.0000 UP_SEQ_FEATURE glycosylation site: N-linked (GlcNAc . . . ) 781 33.4 0.0000 1.6 0.0000 SP_PIR_KEYWORDS disulfide bond 571 24.4 0.0000 1.7 0.0000 UP_SEQ_FEATURE disulfide bond 553 23.6 0.0000 1.7 0.0000 GOTERM_CC_FAT GO:0044421-extracellular region part 285 12.2 0.0000 2.2 0.0000 SP_PIR_KEYWORDS Secreted 376 16.1 0.0000 1.9 0.0000 GOTERM_CC_FAT GO:0005576-extracellular region 444 19.0 0.0000 1.7 0.0000 GOTERM_CC_FAT GO:0005615-extracellular space 180 7.7 0.0000 1.9 0.0000 Annotation Cluster 2 Enrichment Score: 39.79319232227263 GOTERM_BP_FAT GO:0007155-cell adhesion 233 10.0 0.0000 2.5 0.0000 GOTERM_BP_FAT GO:0022610-biological adhesion 233 10.0 0.0000 2.5 0.0000 SP_PIR_KEYWORDS cell adhesion 145 6.2 0.0000 2.8 0.0000 Annotation Cluster 3 Enrichment Score: 28.110221475114905 GOTERM_CC_FAT GO:0005578-proteinaceous extracellular matrix 129 5.5 0.0000 2.9 0.0000 SP_PIR_KEYWORDS extracellular matrix 103 4.4 0.0000 3.5 0.0000 GOTERM_CC_FAT GO:0031012-extracellular matrix 134 5.7 0.0000 2.8 0.0000 GOTERM_CC_FAT GO:0044420-extracellular matrix part 57 2.4 0.0000 3.5 0.0000 Annotation Cluster 4 Enrichment Score: 25.22234604850097 GOTERM_BP_FAT GO:0009611-response to wounding 177 7.6 0.0000 2.5 0.0000 GOTERM_BP_FAT GO:0006954-inflammatory response 110 4.7 0.0000 2.6 0.0000 GOTERM_BP_FAT GO:0006952-defense response 165 7.1 0.0000 2.1 0.0000 Annotation Cluster 5 Enrichment Score: 15.441501146605358 GOTERM_BP_FAT GO:0001568-blood vessel development 88 3.8 0.0000 2.7 0.0000 GOTERM_BP_FAT GO:0001944-vasculature development 89 3.8 0.0000 2.7 0.0000 GOTERM_BP_FAT GO:0048514-blood vessel morphogenesis 72 3.1 0.0000 2.6 0.0000 GOTERM_BP_FAT GO:0001525-angiogenesis 53 2.3 0.0000 2.7 0.0000 Annotation Cluster 6 Enrichment Score: 14.094253321991394 GOTERM_CC_FAT GO:0044459-plasma membrane part 444 19.0 0.0000 1.5 0.0000 GOTERM_CC_FAT GO:0031226-intrinsic to plasma membrane 250 10.7 0.0000 1.5 0.0000 GOTERM_CC_FAT GO:0005887-integral to plasma membrane 242 10.3 0.0000 1.5 0.0000 Annotation Cluster 7 Enrichment Score: 13.217589093630473 GOTERM_BP_FAT GO:0030198-extracellular matrix organization 48 2.1 0.0000 3.5 0.0000 GOTERM_BP_FAT GO:0043062-extracellular structure organization 61 2.6 0.0000 2.8 0.0000 GOTERM_BP_FAT GO:0030199-collagen fibril organization 21 0.9 0.0000 5.5 0.0000 Annotation Cluster 8 Enrichment Score: 11.92555541984095 GOTERM_MF_FAT GO:0030247-polysaccharide binding 59 2.5 0.0000 2.9 0.0000 GOTERM_MF_FAT GO:0001871-pattern binding 59 2.5 0.0000 2.9 0.0000 GOTERM_MF_FAT GO:0005539-glycosaminoglycan binding 55 2.4 0.0000 3.0 0.0000 GOTERM_MF_FAT GO:0030246-carbohydrate binding 99 4.2 0.0000 2.1 0.0000 GOTERM_MF_FAT GO:0008201-heparin binding 41 1.8 0.0000 3.0 0.0000 SP_PIR_KEYWORDS heparin-binding 25 1.1 0.0000 3.1 0.0004 Annotation Cluster 9 Enrichment Score: 11.186259107509017 GOTERM_BP_FAT GO:0006928-cell motion 128 5.5 0.0000 2.0 0.0000 GOTERM_BP_FAT GO:0016477-cell migration 80 3.4 0.0000 2.2 0.0000 GOTERM_BP_FAT GO:0051674- localization of cell 82 3.5 0.0000 2.0 0.0000 GOTERM_BP_FAT GO:0048870-cell motility 82 3.5 0.0000 2.0 0.0000 Annotation Cluster 10 Enrichment Score: 10.235463831336938 SP_PIR_KEYWORDS membrane 930 39.8 0.0000 1.3 0.0000 UP_SEQ_FEATURE topological domain: Extracellular 445 19.0 0.0000 1.4 0.0000 UP_SEQ_FEATURE topological domain: Cytoplasmic 535 22.9 0.0000 1.4 0.0000 SP_PIR_KEYWORDS transmembrane 697 29.8 0.0000 1.2 0.0000 UP_SEQ_FEATURE transmembrane region 693 29.6 0.0000 1.2 0.0000 GOTERM_CC_FAT GO:0031224-intrinsic to membrane 778 33.3 0.0019 1.1 2.7902 GOTERM_CC_FAT GO:0016021-integral to membrane 740 31.6 0.0120 1.1 16.1249

TABLE 28 Top 9 Annotation clusters found enriched among genes negatively correlating with PC1.EMT score (DAVID DB) Fold Category Term Count % PValue Enrichment FDR Annotation Cluster 1 Enrichment Score: 14.055636116126923 SP_PIR_KEYWORDS mitochondrian 148 11.1 0.000 2.7 0.0000 GOTERM_CC_FAT GO:0005739-mitochondrion 172 12.9 0.000 2.3 0.0000 GOTERM_CC_FAT GO:0044429-mitochondrial part 111 8.3 0.000 2.7 0.0000 SP_PIR_KEYWORDS transit peptide 91 6.8 0.000 2.9 0.0000 UP_SEQ_FEATURE transit peptide: Mitochondrion 90 6.8 0.000 2.9 0.0000 GOTERM_CC_FAT GO:0031980-mitochondrial lumen 60 4.5 0.000 3.8 0.0000 GOTERM_CC_FAT GO:0005759-mitochondrial matrix 60 4.5 0.000 3.8 0.0000 GOTERM_CC_FAT GO:0019866-organelle inner membrane 59 4.4 0.000 2.6 0.0000 GOTERM_CC_FAT GO:0005740-mitochondrial envelope 67 5.0 0.000 2.3 0.0000 GOTERM_CC_FAT GO:0005743-mitochondrial inner membrane 54 4.1 0.000 2.5 0.0000 GOTERM_CC_FAT GO:0031966-mitochondrial membrane 63 4.7 0.000 2.3 0.0000 GOTERM_CC_FAT GO:0031967-organelle envelope 78 5.9 0.000 1.8 0.0005 GOTERM_CC_FAT GO:0031975-envelope 78 5.9 0.000 1.8 0.0006 GOTERM_CC_FAT GO:0031090-organelle membrane 115 8.6 0.000 1.5 0.0990 SP_PIR_KEYWORDS mitochondrion inner membrane 30 2.3 0.000 2.3 0.0522 Annotation Cluster 2 Enrichment Score: 8.18712581462025 SP_PIR_KEYWORDS oxidoreductase 79 5.9 0.000 2.1 0.0000 GOTERM_BP_FAT GO:0055114-oxidation reduction 85 6.4 0.000 2.0 0.0000 GOTERM_MF_FAT GO:0009055-electron carrier activity 38 2.9 0.000 2.5 0.0008 Annotation Cluster 3 Enrichment Score: 7.53962677068125 GOTERM_CC_FAT GO:0005759-mitochondrial matrix 60 4.5 0.000 3.8 0.0000 GOTERM_CC_FAT GO:0031980-mitochondrial lumen 60 4.5 0.000 3.8 0.0000 GOTERM_CC_FAT GO:0070013-intracellular organelle lumen 155 11.6 0.002 1.2 2.6842 GOTERM_CC_FAT GO:0031974-membrane-enclosed lumen 160 12.0 0.002 1.2 3.2888 GOTERM_CC_FAT GO:0043233-organelle lumen 155 11.6 0.005 1.2 6.3798 GOTERM_CC_FAT GO:0031981-nuclear lumen 85 6.4 0.974 0.8 100.0000 Annotation Cluster 8 Enrichment Score: 3.2818754734384727 GOTERM_MF_FAT GO:0050662-coenzyme binding 32 2.4 0.000 2.5 0.0039 SP_PIR_KEYWORDS FAD 19 1.4 0.001 2.5 0.8215 GOTERM_MF_FAT GO:0050660-FAD binding 14 1.1 0.001 2.8 1.9608 SP_PIR_KEYWORDS Flavoprotein 17 1.3 0.002 2.4 2.9944 UP_SEQ_FEATURE nucleotide phosphate-binding region: FAD 12 0.9 0.002 2.9 3.9729 UP_SEQ_FEATURE binding site: FAD 7 0.5 0.003 4.7 4.6852 Annotation Cluster 9 Enrichment Score: 2.851945270321666 GOTERM_BP_FAT GO:0006399-tRNA metabolic process 22 1.7 0.000 2.8 0.0532 GOTERM_BP_FAT GO:0034660-ncRNA metabolic process 32 2.4 0.000 2.1 0.2343 GOTERM_BP_FAT GO:0008033-tRNA processing 15 1.1 0.000 3.0 0.7601 SP_PIR_KEYWORDS trna processing 13 1.0 0.001 2.9 2.1777 GOTERM_BP_FAT GO:0034470-ncRNA processing 23 1.7 0.006 1.8 10.9528 GOTERM_BP_FAT GO:0006396-RNA processing 38 2.9 0.493 1.0 99.9995 Annotation Cluster 4 Enrichment Score: 5.213585990413278 GOTERM_BP_FAT GO:0051186-cofactor metabolic process 35 2.6 0.000 2.7 0.0003 GOTERM_BP_FAT GO:0006732-coenzyme metabolic process 29 2.2 0.000 2.8 0.0015 GOTERM_BP_FAT GO:0051188-cofactor biosynthetic process 19 1.4 0.000 2.9 0.1099 GOTERM_BP_FAT GO:0009108-coenzyme biosynthetic process 15 1.1 0.000 3.3 0.2645 Annotation Cluster 5 Enrichment Score: 5.180163320241737 SP_PIR_KEYWORDS nad 34 2.6 0.000 2.7 0.0005 UP_SEQ_FEATURE nucleotide phosphate-binding region: NAD 18 1.4 0.000 3.5 0.0163 UP_SEQ_FEATURE binding site: NAD 13 1.0 0.000 3.9 0.1612 Annotation Cluster 6 Enrichment Score: 4.169776745163271 GOTERM_CC_FAT GO:0005777-peroxisome 29 2.2 0.000 4.0 0.0000 GOTERM_CC_FAT GO:0042579-microbody 29 2.2 0.000 4.0 0.0000 SP_PIR_KEYWORDS peroxisome 25 1.9 0.000 4.1 0.0000 UP_SEQ_FEATURE short sequence motif: Microbody targeting 10 0.8 0.001 3.6 2.3847 signal GOTERM_CC_FAT GO:0044438-microbody part 10 0.8 0.002 3.3 3.4172 GOTERM_CC_FAT GO:0044439-peroxisomal part 10 0.8 0.002 3.3 3.4172 GOTERM_CC_FAT GO:0005778-peroxisomal membrane 7 0.5 0.018 3.2 23.3418 GOTERM_CC_FAT GO:0031903-microbody membrane 7 0.5 0.018 3.2 23.3418 GOTERM_CC_FAT GO:0031907-microbody lumen 4 0.3 0.069 4.1 63.9846 GOTERM_CC_FAT GO:0005782-peroxisomal matrix 4 0.3 0.069 4.1 63.9846 Annotation Cluster 7 Enrichment Score: 3.5116212942526577 GOTERM_MF_FAT GO:0031406-carboxylic acid binding 24 1.8 0.000 2.4 0.2300 GOTERM_MF_FAT GO:0005504-fatty acid binding 11 0.8 0.000 4.2 0.3148 GOTERM_MF_FAT GO:0033293-monocarboxylic acid binding 12 0.9 0.001 3.2 1.6436

GSEA was performed also using gene sets obtained from the MSig database (DB) [Subramanian, A, et al. (2005). Proc. Natl. Acad. Sci 102:15545-15550] (MSigDB) which includes C2 (curated gene sets—Chemical and Genetic Perturbations, Biocarta and KEGG), C3 transcription factors, C5 GO biological process terms, C6 (Oncogenic signature) and C7 (immunologic signatures). The analysis was done using “Romer” algorithm (similar to Gene Set Enrichment Analysis (GSEA)) and the same linear model used to identify genes correlating with PC1.EMT score. The p values obtained across the 6 datasets were merged using Fisher method. Tables 29 to 42 list the top 5 signatures found to be positively or negatively correlated with PC1.EMT within each of the tested Msig.DB.

TABLE 29 Top 10 Chemical and Genetic Perturbations found enriched among genes positively correlating with PC1.EMT score (MSig.DB) S num. p p. value p. adj CHARAFE_BREAST_CANCER_LUMINAL_VS_BASAL_DN 82.89 6 0.00 0.00 CHARAFE_BREAST_CANCER_LUMINAL_VS_MESENCHYMAL_DN 82.89 6 0.00 0.00 TONKS_TARGETS_OF_RUNX1_RUNX1T1_FUSION_ERYTHROCYTE_UP 82.89 6 0.00 0.00 KIM_WT1_TARGETS_8HR_UP 82.89 6 0.00 0.00 DELVS_THYROID_CANCER_UP 82.89 6 0.00 0.00 CASTELLANO_NRAS_TARGETS_UP 82.89 6 0.00 0.00 GAUSSMANN_MLL_AF4_FUSION_TARGETS_F_UP 82.89 6 0.00 0.00 FRIDMAN_SENESCENCE_UP 82.89 6 0.00 0.00 ROZANOV_MMP14_TARGETS_UP 82.89 6 0.00 0.00 PETROVA_ENDOTHELIUM_LYMPHATIC_VS_BLOOD_DN 82.89 6 0.00 0.00

TABLE 30 Top 10 Chemical and Genetic Perturbations found enriched among genes negatively correlating with PC1.EMT score (MSig.DB) S num. p p. value p. adj WALLACE_PROSTATE_CANCER_UP 71.41 6 0.00 0.00 WATANABE_COLON_CANCER_MSI_VS_MSS_DN 66.08 6 0.00 0.00 SERVITJA_ISLET_HNF1A_TARGETS_DN 62.72 6 0.00 0.00 SANSOM_APC_TARGETS_UP 61.54 6 0.00 0.00 ZHOU_PANCREATIC_EXOCRINE_PROGENITOR 58.18 6 0.00 0.00 STEIN_ESRRA_TARGETS_UP 56.70 6 0.00 0.00 BURTON_ADIPOGENESIS_5 55.36 6 0.00 0.00 LE_NEURONAL_DIFFERENTIATION_DN 55.23 6 0.00 0.00 JEON_SMAD6_TARGETS_DN 55.01 6 0.00 0.00 LIEN_BREAST_CARCINOMA_METAPLASTIC_VS_DUCTAL_DN 53.98 6 0.00 0.00

TABLE 31 Top 10 Biocarta pathways found enriched among genes positively correlating with PC1.EMT score (MSig.DB) S num. p p. value p. adj BIOCARTA_LAIR_PATHWAY 59.63 6 0.00 0.00 BIOCARTA_CLASSIC_PATHWAY 58.59 6 0.00 0.00 BIOCARTA_EPHA4_PATHWAY 55.32 6 0.00 0.00 BIOCARTA_COMP_PATHWAY 54.60 6 0.00 0.00 BIOCARTA_MONOCYTE_PATHWAY 52.03 6 0.00 0.00 BIOCARTA_GRANULOCYTES_PATHWAY 50.87 6 0.00 0.00 BIOCARTA_INTEGRIN_PATHWAY 48.92 6 0.00 0.00 BIOCARTA_LYM_PATHWAY 48.22 6 0.00 0.00 BIOCARTA_VITCB_PATHWAY 45.75 6 0.00 0.00 BIOCARTA_FIBRINOLYSIS_PATHWAY 45.37 6 0.00 0.00

TABLE 32 Top 10 Biocarta pathways found enriched among genes negatively correlating with PC1.EMT score (MSig.DB) S num. p p. value p. adj BIOCARTA_MITOCHONDRIA_PATHWAY 42.23 6 0.00 0.01 BIOCARTA_RANMS_PATHWAY 42.16 6 0.00 0.01 BIOCARTA_PTC1_PATHWAY 41.78 6 0.00 0.01 BIOCARTA_ATRBRCA_PATHWAY 35.39 6 0.00 0.09 BIOCARTA_NUCLEARRS_PATHWAY 34.73 6 0.00 0.11 BIOCARTA_CELLCYCLE_PATHWAY 33.34 6 0.00 0.18 BIOCARTA_G2_PATHWAY 32.93 6 0.00 0.21 BIOCARTA_CASPASE_PATHWAY 31.86 6 0.00 0.31 BIOCARTA_MOM_PATHWAY 31.75 6 0.00 0.33 BIOCARTA_G1_PATHWAY 29.41 6 0.00 0.74

TABLE 33 Top 10 KEGG pathways found enriched among genes positively correlating with PC1.EMT score (MSig.DB) S num. p p. value p. adj KEGG_AXON_GUIDANCE 78.73 6 0.00 0.00 KEGG_FOCAL_ADHESION 78.73 6 0.00 0.00 KEGG_ECM_RECEPTOR_INTERACTION 78.73 6 0.00 0.00 KEGG_GLYCOSAMINOGLYCAN_BIOSYNTHESIS_CHONDROITIN_SULFATE 75.07 6 0.00 0.00 KEGG_REGULATION_OF_ACTIN_CYTOSKELETON 71.57 6 0.00 0.00 KEGG_LEUKOCYTE_TRANSENDOTHELIAL_MIGRATION 70.00 6 0.00 0.00 KEGG_CELL_ADHESION_MOLECULES_CAMS 67.30 6 0.00 0.00 KEGG_PATHWAYS_IN_CANCER 67.17 6 0.00 0.00 KEGG_COMPLEMENT_AND_COAGULATION_CASCADES 63.45 6 0.00 0.00 KEGG_HYPERTROPHIC_CARDIOMYOPATHY_HCM 61.53 6 0.00 0.00

TABLE 34 Top 10 KEGG pathways found enriched among genes negatively correlating with PC1.EMT score (MSig.DB) S num. p p. value p. adj KEGG_PEROXISOME 64.62 6 0.00 0.00 KEGG_SELENOAMINO_ACID_METABOLISM 63.30 6 0.00 0.00 KEGG_BUTANOATE_METABOLISM 60.53 6 0.00 0.00 KEGG_CITRATE_CYCLE_TCA_CYCLE 60.25 6 0.00 0.00 KEGG_TRYPTOPHAN_METABOLISM 55.30 6 0.00 0.00 KEGG_VALINE_LEUCINE_AND_ISOLEUCINE_BIOSYNTHESIS 52.23 6 0.00 0.00 KEGG_PYRUVATE_METABOLISM 51.60 6 0.00 0.00 KEGG_MATURITΥ_ONSET_DIABETES_OF_THE_YOUNG 50.94 6 0.00 0.00 KEGG_ARGININE_AND_PROLINE_METABOLISM 49.03 6 0.00 0.00 KEGG_VALINE_LEUCINE_AND_ISOLEUCINE_DEGRADATION 47.23 6 0.00 0.00

TABLE 35  Top 10 transcription factor targets found enriched among genes positively correlating with PC1.EMT score (MSig.DB) S num.P p.value p.adj V$AP1_Q6_01 82.89 6 0.00 0.00 V$AP1_Q4_01 82.89 6 0.00 0.00 TGANTCA_V$AP1_C 82.89 6 0.00 0.00 V$AML1_01 81.51 6 0.00 0.00 V$AML1_Q6 81.51 6 0.00 0.00 GGGTGGRR_V$PAX4_03 81.51 6 0.00 0.00 V$AP1_C 80.70 6 0.00 0.00 V$STAT5B_01 80.70 6 0.00 0.00 V$NFKB_Q6 80.12 6 0.00 0.00 CAGCTG_V$AP4_Q5 79.67 6 0.00 0.00

TABLE 36  Top 10 transcription factor targets found enriched among genes positively correlating with PC1.EMT score (MSig.DB) S num.P p.value p.adj V$MYCMAX_01 56.53 6 0.00 0.00 V$PPARG_01 44.02 6 0.00 0.01 V$E2F1_Q6_01 40.53 6 0.00 0.04 V$E2F_Q4_01 40.43 6 0.00 0.04 V$E2F1_Q4_01 40.17 6 0.00 0.04 SGCGSSAAA_V$E2F1DP2_01 39.82 6 0.00 0.05 V$E2F_Q6_01 38.66 6 0.00 0.07 V$E2F_Q3 38.63 6 0.00 0.07 V$E2F1_Q3 38.54 6 0.00 0.08 V$HNF4_01 38.20 6 0.00 0.09

TABLE 37 Top 10 GO Biological Process terms found enriched among genes positively correlating with PC1.EMT score (MSig.DB) S num. p p. value p. adj TISSUE_DEVELOPMENT 82.89 6 0.00 0.00 ORGAN_DEVELOPMENT 81.51 6 0.00 0.00 CELL_SUBSTRATE_ADHESION 78.29 6 0.00 0.00 CELL_MATRIX_ADHESION 77.92 6 0.00 0.00 REGULATION_OF_CELL_GROWTH 77.92 6 0.00 0.00 REGULATION_OF_BIOLOGICAL_QUALITY 77.48 6 0.00 0.00 MULTICELLULAR_ORGANISMAL_DEVELOPMENT 77.48 6 0.00 0.00 ANATOMICAL_STRUCTURE_DEVELOPMENT 77.11 6 0.00 0.00 CELL_MIGRATION 76.54 6 0.00 0.00 NEGATIVE_REGULATION_OF_GROWTH 76.23 6 0.00 0.00

TABLE 38 Top 10 GO Biological Process terms found enriched among genes negatively correlating with PC1.EMT score (MSig.DB) S num. p p. value p. adj ORGANIC_ACID_METABOLIC_PROCESS 48.24 6 0.00 0.00 COENZYME_BIOSYNTHETIC_PROCESS 46.92 6 0.00 0.00 CARBOXYLIC_ACID_METABOLIC_PROCESS 45.86 6 0.00 0.01 RRNA_METABOLIC_PROCESS 42.57 6 0.00 0.02 RIBOSOME_BIOGENESIS_AND_ASSEMBLY 41.80 6 0.00 0.03 ENERGY_DERIVATION_BY_OXIDATION_OF_ORGANIC_COMPOUNDS 40.96 6 0.00 0.04 CELLULAR_PROTEIN_COMPLEX_DISASSEMBLY 40.52 6 0.00 0.05 RRNA_PROCESSING 40.44 6 0.00 0.05 GLYCOPROTEIN_BIOSYNTHETIC_PROCESS 39.42 6 0.00 0.07 MACROMOLECULE_BIOSYNTHETIC_PROCESS 39.27 6 0.00 0.08

TABLE 39 Top 10 Oncogenic signatures found enriched among genes positively correlating with PC1.EMT score (MSig.DB) S num. p p. value p. adj ESC_V6.5_UP_EARLY.V1_DN 82.89 6 0.00 0.00 CAHOY_ASTROGLIAL 82.89 6 0.00 0.00 PTEN_DN.V2_UP 81.51 6 0.00 0.00 BMI1_DN.V1_UP 80.70 6 0.00 0.00 BMI1_DN_MEL18_DN.V1_UP 79.31 6 0.00 0.00 RPS14_DN.V1_UP 78.73 6 0.00 0.00 ATF2_S_UP.V1_DN 77.92 6 0.00 0.00 TGFB_UP.V1_UP 77.35 6 0.00 0.00 MEL18_DN.V1_DN 77.11 6 0.00 0.00 KRAS.DF.V1_UP 77.11 6 0.00 0.00

TABLE 40 Top 10 Oncogenic signatures found enriched among genes negatively correlating with PC1.EMT score (MSig.DB) S num. p p. value p. adj MYC_UP.V1_UP 57.05 6 0.00 0.00 RPS14_DN.V1_DN 49.57 6 0.00 0.00 STK33_NOMO_DN 49.13 6 0.00 0.00 RB_DN.V1_UP 46.95 6 0.00 0.00 STK33_DN 43.77 6 0.00 0.00 GCNP_SHH_UP_LATE.V1_UP 38.67 6 0.00 0.02 E2F1_UP.V1_UP 37.70 6 0.00 0.03 PRC2_EZH2_UP.V1_UP 37.46 6 0.00 0.04 EIF4E_UP 32.51 6 0.00 0.22 RB_P130_DN.V1_UP 32.13 6 0.00 0.25

TABLE 41 Top 10 immunologic signatures found enriched among genes positively correlating with PC1.EMT score (MSig.DB) S num. p p. value p. adj GSE1432_6H_VS_24H_IFNG_MICROGLIA_UP 82.89 6 0.00 0.00 GSE14350_IL2RB_KO_VS_WT_TREG_DN 82.89 6 0.00 0.00 GSE17721_12H_VS_24H_LPS_BMDM_UP 82.89 6 0.00 0.00 GSE24142_EARLY_THYMIC_PROGENITOR_VS_DN3_THYMOCYTE_ADULT_UP 82.89 6 0.00 0.00 GSE24634_TEFF_VS_TCONV_DAY10_IN_CULTURE_DN 82.89 6 0.00 0.00 GSE26495_NAIVE_VS_PD1HIGH_CD8_TCELL_DN 82.89 6 0.00 0.00 GSE3337_CTRL_VS_16H_IFNG_IN_CD8POS_DC_UP 82.89 6 0.00 0.00 GSE360_DC_VS_MAC_T_GONDI1_DN 82.89 6 0.00 0.00 GSE3982_DC_VS_TH1_UP 82.89 6 0.00 0.00 GSE3982_DC_VS_TH2_UP 82.89 6 0.00 0.00

TABLE 42 Top 10 immunologic signatures found enriched among genes negatively correlating with PC1.EMT score (MSig.DB) S num. p p. value p. adj GSE27786_ERYTHROBLAST_VS_MONO_MAC_UP 59.14 6 0.00 0.00 GSE18791_CTRL_VS_NEWCASTLE_VIRUS_DC_8H_UP 56.51 6 0.00 0.00 GSE18791_CTRL_VS_NEWCASTLE_VIRUS_DC_6H_UP 54.69 6 0.00 0.00 GSE27786_LIN_NEG_VS_BCELL_UP 52.38 6 0.00 0.00 GSE27786_LIN_NEG_VS_NEUTROPHIL_UP 52.37 6 0.00 0.00 GSE14308_TH2_VS_INDUCED_TREG_DN 52.07 6 0.00 0.00 GSE18791_CTRL_VS_NEWCASTLE_VIRUS_DC_14H_UP 51.65 6 0.00 0.00 GSE14350_TREG_VS_TEFF_IN_IL2RB_KO_DN 49.37 6 0.00 0.00 GSE18791_CTRL_VS_NEWCASTLE_VIRUS_DC_4H_UP 49.23 6 0.00 0.00 GSE18791_CTRL_VS_NEWCASTLE_VIRUS_DC_10H_UP 48.84 6 0.00 0.00

PC1.EMT score expression was also compared with a set of 75 gene signatures designed to capture some biological functions. Those signatures were obtained from different sources (databases, literature, etc.). The correlation coefficients were combined using DerSimonian-Laird (DSL) meta-analytic method. FIG. 11 is a forest plot showing correlation between PC1.EMT score intensity and relevant gene signatures. Only signatures showing an absolute estimated coefficient above 0.3 and an adjusted p value bellow 0.05 were plotted.

The enrichment analysis evidenced that PC1.EMT is still strongly associated with EMT. For instance, response to wounding, cell motility, extracellular matrix remodeling, activation of TGFbeta signalling, angiogenesis are all well known phenomena associated with EMT. Activation of Notch signaling was also observed, which has been also suggested to be involved in EMT.

The role of WNT signalling in EMT has been also described in literature. However, contradictory results were observed with different WNT signatures showing different behavior (some positively and other negatively correlated). This is also in line with the APC mutations results, where it was observed that only specific mutations were showing lower PC1.EMT score compare to WT.

Concerning the negatively correlated features, there was a clear effect in the mitochondrial metabolism and function. Activation of MYC was also inversely correlated with PC1.EMT score.

PC1.EMT and Clinico-Pathological-Molecular Features

The expression of PC1.EMT score was also compared with the available Clinico-Pathological-Molecular features for each datasets. FIG. 12A to 12U are boxplots of PC1.EMT score split by the clinico-pathological-molecular features age (FIG. 12A), gender (FIG. 12B), stage (FIG. 12C), T.stage (FIG. 12D), N.stage (FIG. 12E), grade (FIG. 12F), adj. treatment (FIG. 12G), lymphovascular invation LVI (FIG. 12H), perineural invasion PNI (FIG. 12I), bowel obstruction (FIG. 12J), positive margin (FIG. 12K), mucinous (FIG. 12L), tumor site (FIG. 12M), left or right tumor site (FIG. 12N), MSI (FIG. 12O), PIK3Ca (FIG. 12P), TP53 (FIG. 12Q), LOH18 (FIG. 12R), BRAF (FIG. 12S), PIK3Ca (FIG. 12T), and TYMS using the PETACC dataset.

FIG. 13A to 13G are boxplots of PC1.EMT score split by the clinico-pathological-molecular features age (FIG. 13A), gender (FIG. 13B), stage (FIG. 13C), T.stage (FIG. 13D), mucinous (FIG. 13E), tumor site (FIG. 13F), and left or right tumor site (FIG. 13G) using the ALMAC dataset.

FIG. 14A to 14K are boxplots of PC1.EMT score split by the clinico-pathological-molecular features age (FIG. 14A), gender (FIG. 14B), stage (FIG. 14C), KRAS (FIG. 14D), MSI (FIG. 14E), KRAS (FIG. 14F), BRAF (FIG. 14G), TP53 (FIG. 14H), CIMP (FIG. 14I), adj. treatment (FIG. 14J), and CIN (FIG. 14K) using the French dataset.

FIG. 15A to 15D are boxplots of PC1.EMT score split by the clinico-pathological-molecular features age (FIG. 15A), gender (FIG. 15B), Dukes stage (FIG. 15C), and tumor site (FIG. 15D) using the GSE14333 dataset.

FIG. 16A to 16I are boxplots of PC1.EMT score split by the clinico-pathological-molecular features age (FIG. 16A), gender (FIG. 16B), stage (FIG. 16C), T.stage (FIG. 16D), N.stage (FIG. 16E), tumor site (FIG. 16F), left or right tumor site (FIG. 16G), MSI (FIG. 16H), and TP53 (FIG. 16I) using the GEO41258 dataset.

FIGS. 17A to 17L are boxplots of PC1.EMT score split by the clinico-pathological-molecular features age (FIG. 17A), gender (FIG. 17B), stage (FIG. 17C), T.stage (FIG. 17D), lympovascular invation (LVI) (FIG. 17E), perineural invasion (PNI) (FIG. 17F), mucinous (FIG. 17G), tumor site (FIG. 17H), left or right tumor site (FIG. 17I), MSI (FIG. 17J), KRAS (FIG. 17K), and BRAF (FIG. 17J) using the TCGA dataset.

PC1.EMT high score was usually associated with higher T and N stages, higher grade, mucinous histology. It was also higher in the right sided, MSI-High, BRAF V600E mutants and CIMP positive.

PC1.EMT and Copy Number Variations (CNVs)

In order to assess if PC1.EMT is or not correlating with chromosomal instability (CIN), TCGA samples were split based on the number of chromosomal rearrangement observed. The CIN status was assigned according to CGH alteration profile. A CIN rate was designed as the proportion of chromosomes showing gain (segmented ratio>0.5) or loss (segmented ratio<−0.5) events (excluding sex chromosomes). A tumor having an alteration rate superior to 10% was considered CIN+, otherwise CIN−. FIG. 18A to 18B are boxplot of the PC1.EMT score split by chromosomal instability (CIN) status in TCGA (FIG. 18A) and French (FIG. 18B) datasets. PC1.EMT high score was associated with lower chromosomal instability.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. 

1. A method for predicting the recurrence of colorectal cancer in a human patient, comprising: (a) assaying colorectal cells obtained from the human patient for the expression level of ten (10) or more genes listed in TABLE 2A, or their corresponding expression products, and ten (10) or more genes listed in TABLE 2B, or their corresponding expression products, and using normalized values of the expression levels to calculate a colorectal cancer (CRC) score; (b) assaying colorectal cells obtained from the human patient for the expression levels of ten (10) or more genes listed in TABLE 1A, or their corresponding expression products, and ten (10) or more genes listed TABLE 1B, or their corresponding expression products, and using normalized values of the expression levels to calculate an Epithelial to Mesenchymal Transition (EMT) score; and (c) calculating the difference between the CRC score and the EMT score to arrive at a Recurrence Signature Score that predicts risk of colorectal cancer recurrence.
 2. The method of claim 1, further comprising displaying or outputting to a user, user interface device, computer readable storage medium, or local or remote computer system the calculated risk of colorectal cancer recurrence.
 3. The method of claim 1, further comprising treating the human patient with post-operative adjuvant chemotherapy if a high risk of colorectal cancer recurrence is determined.
 4. The method of claim 3, wherein the chemotherapy comprises a 5-fluorouracil (5-FU) therapy.
 5. The method of claim 1, wherein increased expression of the genes listed in TABLE 2A, or their corresponding expression products, increases the CRC score; and wherein increased expression of the genes listed in TABLE 2B, or their corresponding products, decreases the CRC score.
 6. The method of claim 1, wherein increased expression of the genes listed in TABLE 1A, or their corresponding expression products, increases the EMT score; and wherein increased expression of the genes listed in TABLE 1B, or their corresponding products, decreases the EMT score.
 7. The method of claim 1, wherein the expression levels are normalized by quantile normalization.
 8. The method of claim 1, wherein the normalized expression values of the expression levels for each assayed gene are given equal weight in calculating the CRC score.
 9. The method of claim 1, wherein the normalized expression values of the expression levels for each assayed gene are given equal weight in calculating the EMT score.
 10. The method of claim 1, wherein the colorectal cancer comprises Dukes B (stage II) or Dukes C (stage III) colorectal cancer.
 11. A method for analyzing a colorectal cancer tissue sample to determine adjuvant chemotherapy is needed to prevent colorectal cancer recurrence in a human patient, the method comprising: (a) assaying colorectal cells obtained from the human patient for normalized expression values of ten (10) or more genes listed in TABLE 1A, ten (10) or more genes listed in TABLE 1B, ten (10) or more genes listed in TABLE 2A, and ten (10) or more genes listed in TABLE 2B; and (b) inputting the normalized expression values into a computer programmed to execute an algorithm to convert the normalized expression values to a Recurrence Signature Score indicative of a likelihood of the risk of colorectal cancer recurrence, wherein the algorithm gives reduced weight to the normalized expression values for genes that are listed in more than one of TABLE 1A, TABLE 1B, TABLE 1C, and TABLE 1D.
 12. The method of claim 11, wherein the algorithm converts the normalized expression values for the genes listed in TABLE 2A and TABLE 2B to a colorectal cancer (CRC) score, wherein the algorithm converts the normalized expression values for the genes listed in TABLE 1A and TABLE 1B to an Epithelial to Mesenchymal Transition (EMT) score, wherein the Recurrence Signature Score is determined by calculating the difference between the CRC score and the EMT score.
 13. The method of claim 12, wherein increased expression of the genes listed in TABLE 2A, or their corresponding expression products, increases the CRC score; and wherein increased expression of the genes listed in TABLE 2B, or their corresponding products, decreases the CRC score.
 14. The method of claim 12, wherein increased expression of the genes listed in TABLE 1A, or their corresponding expression products, increases the EMT score; and wherein increased expression of the genes listed in TABLE 1B, or their corresponding products, decreases the EMT score.
 15. The method of claim 11, further comprising treating the human patient with post-operative adjuvant chemotherapy if a high risk of colorectal cancer recurrence is determined.
 16. The method of claim 15, wherein the chemotherapy comprises a 5-fluorouracil (5-FU) therapy.
 17. The method of claim 11, wherein the expression levels are normalized by quantile normalization.
 18. The method of claim 11, wherein the normalized expression values of the expression levels for each assayed gene are given equal weight in calculating the CRC score.
 19. The method of claim 11, wherein the normalized expression values of the expression levels for each assayed gene are given equal weight in calculating the EMT score.
 20. The method of claim 11, wherein the colorectal cancer comprises Dukes B (stage II) or Dukes C (stage III) colorectal cancer. 